Compositions and methods for treating immunological dysfunction

ABSTRACT

The present invention is directed to methods of lymphotherapy to treat cancer, infection and autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/200,999, filed on Jul. 1, 2016, now allowed, which claims the benefitof priority of U.S. Provisional Application No. 62/187,493, filed Jul.1, 2015, the contents of each of which are hereby incorporated byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2016, isnamed COOC-001_001US_Seq_Listing and is 60 KB in size.

FIELD OF THE INVENTION

The present invention relates to immunotherapy. In particular theinvention provides methods of activating and expanding immune cellpopulations ex vivo and the use of the cells to treat immunologicaldysfunctions such as cancer, infection and autoimmune disorders.

BACKGROUND OF THE INVENTION

The immune system is designed to eradicate a large number of pathogens,as well as tumors, with minimal immunopathology. When the immune systembecomes defective, however, numerous disease states result.Immunotherapy is a treatment modality that seeks to harness the power ofthe human immune system to treat disease.

One immunotherapy method is a type of cell therapy called adoptiveimmunotherapy. Adoptive immunotherapy is a cell therapy that involvesthe removal of immune cells from a subject, the ex-vivo processing(i.e., activation, purification and/or expansion of the cells) and thesubsequent infusion of the resulting cells back into the same ordifferent subject. Adoptive immunotherapy, which involves the transferof autologous antigen-specific T cells generated ex vivo, is a promisingstrategy to treat cancer.

There is a need for cancer immunotherapy that provides protective andtherapeutic immunity to a wide variety of tumor types. Current, adoptiveimmunotherapy treatments have infrequent and sporadic efficacy. Thus,there is a need to identify and solve these problems in order toincrease the efficacy of adoptive immunotherapy protocols.

SUMMARY OF THE INVENTION

In various aspects the invention provides a ghost cell trainer (GHT)with a plasma membrane having a plurality of major histocompatibilitycomplex (MHC) molecules; and a plurality of a protein of interest. Theprotein is glycosylphosphatidylinositol (GPI) anchored on the cellsurface.

The invention also provides a ghost cell (GHC), having the followingcharacteristics, is substantially free of all intracellular componentsand a plasma membrane having a plurality of MHC molecules and aplurality of a protein of interest. The protein is GPI anchored on thecell surface.

In a further aspect the invention provides a method of producing a ghostcell (GHC) by lysing the GHT according to the invention to form a cellthat is substantially free of all intracellular components.

In yet another aspect the invention provides a method of producing a GHCby transfecting a cell expressing an MHC molecule with one or moreplasmids containing a gene encoding a protein of interest and a GPIsignaling anchor such that the protein of interest is expressed and GPIanchored to the cell surface; and lysing the cells of to form a cellthat is substantially free of all intracellular components.

In another aspect the invention provides methods of producing a GHC bytransfecting a cell with a plasmid encoding an MHC protein and a GPIsignaling anchor; one or more plasmids comprising a gene encoding aprotein of interest and a GPI signaling anchor such that the MHCmolecule and the protein of interest are expressed and GPI anchored tothe cell surface. The cell is then lysed as to form a cell that issubstantially free of all intracellular components.

The GPI anchored protein of interest and/or the MHC molecules is withina lipid raft.

MHC molecules include for example HLA-Cw*3 and/or HLA-Cw*5.

The protein of interest is one or more co-stimulatory molecules orcytokines such as CD80, CD86, CD137, IL-15, CD28, IL-21, IL-2,IFN-gamma, IL-10, IL-12, TGF-beta, CD40L, IL-4, or CD64. In some aspectsthe co-stimulatory molecule is CD64 and the CD64 is loaded with anantibody such as an antibody that is specific for NK2GD or CD3.

Lysing is accomplished for example by pH, temperature, chemical,physical, hypotonic or hypertonic means.

Also provided is the GHT or the GHC or population thereof produced bythe methods of the invention.

In yet another aspect the invention provides methods of expanding anoligoclonal immune cell population by co-culturing the GHC according tothe invention with a population of immune cells. Also provided aremethods of expanding an oligoclonal NK-cell population by co-culturingthe GHC according to the invention wherein the GHC expresses CD64 loadedwith an anti-NK2GD antibody with a NK-cell.

The immune cell is for example a T-cell, an NK-cell or a B-cell.Optimally, the ratio of GHC to immune cells is about 1:2 to 1:4.Optionally, IL-21, IL-15 IL-2, IL-4, IL-10, IL-12 or any combinationthereof is added to the culture.

Also included in the invention is an expanded oligoclonal immune cellpopulation produced by the method of the invention.

In other aspects the invention provides methods of treating cancer,infection or an autoimmune disease in a subject in need thereof byintravenously or intratumorally administering a first dose of theexpanded immune cell population according to the invention. The canceris a hematologic cancer such as leukemia or lymphoma or a solid cancer.The leukemia is Acute myeloid leukemia (AML), Chronic myeloid leukemia(CIVIL), Acute lymphocytic leukemia (ALL), or Chronic lymphocyticleukemia (CLL). The lymphoma is B-cell lymphoma.

The solid cancer is for example, osteosarcoma, hemangiosarcoma,transitional cell carcinoma, melanoma, glioblastoma, neuroblastoma,mammary carcinoma, or a sarcoma or carcinoma of the gastrointestinalsystem.

Optionally, a second dose of the cell population is administered 7 daysafter the first dose.

In some aspects, a third dose of the cell population is administered 7days after the second dose.

The subject has received a HSCT, high dose Cytoxan therapy, chemotherapyor radiation therapy prior to the administration of the cell population.

In some embodiments of the invention, the cancer is osteosarcoma and thecells are administered at a dose of 5×10⁸ cells/m² 24-72 hours posttargeted or whole body radiation.

In other embodiments of the hematologic cancer is lymphoma and the cellsare administered at a dose of 5×10⁸ cells/m².

In further embodiments, the hematologic cancer is acute myeloid leukemiaand the cell population is administered every 7 to 10 days for a totalof no more than 8 doses. Preferably the does is 2×10⁸ cells/m² to 5×10⁸cells/m².

In other embodiments the cancer is hemangiosarcoma and the cells areadministered at a dose of 5×10⁸ cells/m².

In yet a further aspect of the invention provides methods of treating animmunological dysfunction in a subject in need thereof comprisingadministering at least one dose of the expanded immune cell populationof the invention. Immunological dysfunctions include for example cancer,GVHD, chronic viral infection or an autoimmune disorder.

The subject is in an active cycle of the autoimmune disorder.

The cells are administered at a dose of 1×10⁷ cells/m² to 5×10⁹cells/m².

The subject is a companion animal or a human. The companion animal isfor example a canine, a feline or an equine. The expanded immune cellsare autologous or allogeneic to the subject.

In further aspects, the invention provides a method of producing a tumorantigen specific T-cell population by co-culturing a population ofT-cells that has been transfected with a plasmid containing a nucleicacid encoding activation-induced deaminase (AID) with the GHC accordingto the invention, and a tumor or a tumor cell line.

The plasmid further comprises a promoter, a signaling peptide, a startcodon, a stop codon, a kozak sequence and/or a detectable label. Thepromoter is a cell specific or inducible promoter. The detectable labelis for example a fluorescent protein. Preferably, the plasmid isproduced by DNA printing or through a bacterial process.

The tumor and the T-cells are autologous or allogenic. The T-cells areprimary cells or immortalized cells. The T-cells are human, canine,feline murine or equine.

Optionally, the T-cell population expresses a mutated T-cell receptor(TCR) or a mutated chimeric antigen receptor (CAR). Optionally, themethod further includes sequencing the TCR or CAR. The invention alsoincludes the mutated TCR or CAR sequences of identified by the method ofthe invention as well a T-cell, NK-cell or B-cell population engineeredto express the mutated TCR or CAR sequences. Cells expressing themutated TCR or CAR sequences can be expanded by co-culturing with a GHCaccording to the methods of the invention. These expanded cellpopulation can be used to treat cancer, infection or an autoimmunedisorder by administering to a subject the population of expanded cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows lipid rafts of ghost cells.

FIG. 2A shows T-cell-antigen recognition and the immunological synapse(Johannes B. Huppa & Mark M. Davis, Nature Reviews Immunology 3, 973-983(December 2003)).

FIG. 2B shows lipofectamine vesicles containing gene of interesttransposon and transposase entering the intracellular and nuclearmembranes. The transposase cutting the gene of interest out of thetransposon and randomly inserting it into the pre-ghost cell's DNA.

FIG. 3 shows the creation of ghost cells expressing GPI anchoredproteins on cell membrane lipid rafts using hypotonic solution for acutecell lysis and immediate co-culture with mammalian lymphocytes.

FIG. 4 shows the process of mammalian lymphocytes isolated and expandedon ghost Cells, cryopreserved and re-infused.

FIG. 5 shows the reconstitution of a normal, intact immune system aftertotal body radiation and hematopoietic stem cell transplant to treat amalignancy and the associated tumor-driven dysfunctional and ablatedimmune system.

FIG. 6 is a vector map of the novel generation of TCR and CAR sequencesto be expressed in primary cells. Cell Specific Promoter: CD3z, BCR, NKR(Species specific also) for added safety benefit. The CAR/TCR will onlywork in a specific cell type cannot be transferred. Mutated TCR/CARSequence of choice with transmembrane domain Chimeric Endodomain: CD3z,CD28, 41BB as possible combinations for more potent killing of tumor.Kanamycin selection for clinical vector.

FIG. 7 is a vector map of co-stimulatory molecules, cytokines, andantigens to be anchored with GPI for lipid raft sequestering on theplasma membrane of a ghost cell trainer. CMV: Human CMV promoter; SP:Signaling Peptide for cellular location: Gene of Interest:Co-Stimulatory Molecule**; GPI: CD55 Propeptide and Lipidation GPISignaling Anchor. **This can be interchanged with CD64, CD86, CD137L,and human IL-15. All other regions will constant regardless of the Geneof Interest. This can be used for the ghost cells for NK and T cellexpansion. For NK expansion, anti-NK2GD antibody can be loaded on theCD64 receptor prior to cryopreservation and ghosting.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for inducing asystemic immune response against a tumor or pathogen. In particular, thepresent invention provides methods of immune cell activation (e.g.T-cells, B-cells, or NK-cells) for use in immunotherapy to treatcancers, infection and autoimmune disorders in humans, sport animals andcompanion animals. Activation of the immune cells by the methods of theinvention results in cell expansion.

Biological therapies, such as NK or T-cell therapies, can target diseaseby employing mechanisms independent of chemo-radio-therapies. There isan unmet need to provide companion canines, other companion animals andhumans, with lymphotherapies that can be used in combination withstandard of care therapies or alone. The present invention providestargeted, T-cell receptor (TCR) oligoclonal T-cell, Natural Killer (NK)cell, and other immune cells lymphotherapy that can be utilized againstmalignancies, infection and autoimmune diseases. As a means toaccelerate broader use of targeted lymphotherapy for mammalian, e.g.human use, the present invention provides following: (i) the “ghostcells” to numerically expand CAR-TCR-specific T cells, NK cells andB-cells for clinical use, (ii) the method of cellular (T and NK)HLA-Cw*3 and 5 expansion on ghost cells, (iii) TCR sequences and tumortargets generated from expansion method and patient response, (iv)dosing and conditioning treatment schedules lymphotherapy for treatmentof disease, (v) biomarker and immunoscore useful in screening anddiagnosing, and (vi) genetically modified cellular therapy using TCRsand chimeric antigen receptors (CAR).

The primary obstacles faced by cellular based immunotherapies areovercoming both peripheral and central tolerance issues as the tumorevolves through immunoediting pressures to become unrecognizable andavoidable immunologically and develops the ability to suppress ananti-tumor/pro-inflammatory response. The progression or development ofmalignancies, autoimmune diseases and infection (e.g, chronic viralinfection can produce immunological dysfunction by alterations incytokine production (both locally and systemically), inhibition ofdendritic cell (DC) maturation thereby producing antigen presentationdefects, T-cell dysfunctions characterized by ratio imbalances,signaling issues, gene modulation, immune synaptic malformations, andself-antigen presentation. Tumors can produce strong immunosuppressivecytokines, such as IL-10 and TGF-B.

The tumor micro-environment (TME) is a complex milieu of tumor cells,endothelial cells, stromal cells, tumor associated macrophages (TAM),fibroblasts, myeloid derived suppressor cells (MDSC), NK cells, Tregs,CTLs, and monocytes. All of which are influenced directly or indirectlyby the tumor. Chemo- and radio-therapies have also been shown toincrease the expression of neo-antigens by residual tumor. Otherpre-conditioning regimens alter TH₁/TH₂ profiles, reduce the numbers ofMDSCs, Tregs, and TAMS, activate DC, cause tumor necrotic death forneo-antigen release, aymphotherapy will reverse the immunologicaldysfunction associated with malignancies and autoimmune diseases alike.

In order to accelerate broader use of targeted lymphotherapy, thepresent invention provides a method of producing “ghost cells” tonumerically expand oligoclonal TCR-specific T cells, B-cells and NKcells. Lymphotherapy will reverse the immunological dysfunctionassociated with malignancies, infection and autoimmune diseases alike.

Ghost Cells

As used herein the term “ghost cells” is meant to include animmortalized cell line that has been rendered spherostomatocytic andcontains little or no intracellular components, but retain plasma cellmembrane and lipid raft integrity. The term “ghost cell” results fromthe preparation method to render the cell empty and non-viable.

Ghost cells according to the invention have on their plasma membrane aplurality of MHC molecules, e.g., MHC class I or MHC class II and aplurality of a protein of interest that is glycosylphosphatidylinositol(GPI) anchored in the plasma membrane. The use of GPI anchors is indirect contrast to the current methods that utilize transmembranedomains to anchor proteins in plasma membrane. Anchoring via atransmembrane domain results in variable expression throughout theplasma membrane. In contrast, the present inventor has discovered thatGPI anchoring ensures that all transfected genes are expressed on thecell surface in close proximity inside lipid rafts. This conformationyields superior immune cell expansion. Preferably the ghost cellscontain 10⁵ to 10¹⁰, more preferably 10⁶ to 9×10⁹ lipid rafts containingthe protein of interest.

Ghost cells are prepared by genetically modifying a cell to express aprotein of interest. The protein of interest is anchored to lipid raftsvia a glycosylphosphatidylinositol anchor (GPI) that is added to theprotein during ER post-translational modification. Preferably the cellexpresses endogenous MEW molecules. Alternatively the cell isgenetically modified to express MEW molecules. For example the cellexpresses or has been genetically modified to express HLA-Cw*3 and/orHLA-Cw*5. One skilled in the art will readily recognize the appropriateMHC molecule required to treat a particular disease or disorder.

The genetically modified cells, prior to treatment to become ghost cellsare referred to herein as “ghost cell trainers”.

The cell that is genetically modified to produce the ghost cell traineris preferably a cell of human, canine, equine, murine or feline origin.The cell is a primary cell culture or an immortalized cell line. Forexample, the cell is an adherent or suspended mammalian cell line.Exemplary cells lines suitable for the production of ghost cellstrainers include for example, immortalized cells lines such as K562,722.21 or Jurkat.

Standard flow cytometry techniques are used to determine ghost celltrainer surface expression of lipid rafts, proteins, co-stimulatory, etcin each cell line before lysis. At least 70%, 75%, 80%, 85%, 90%, 95% ormore of the ghost cell trainers in the population must express theprotein of interest in lipid rafts. Ghost cell trainer expression ofmolecules, lipid rafts, etc must be greater than 90% to continue withcryopreservation and creation of ghost cells. To determine theexpression in lipid rafts, as well as, surface placement and relativequantification, standard techniques of antibody patching andimmunofluorescence microscopy is utilized. This allows the visualizationand quantification of lipid raft components on the ghost cell trainersurface at various time points during cell culture. Ghost cell trainersare cultured using standard techniques known in the art for theparticular cell being utilized. Suitable culture conditions for ghostcell trainers are for example 39° C., 5% CO₂, 95% humidified atmosphere;complete media (RPMI/DMEM media), 10-20% fetal calf serum, 5-15%glutamax. Culture media is changed 1 to 5 times per week to maintaincells at 10⁶ cells/mL; in either flasks, bags, or closed systembioreactors. Evaluation of ghost cell trainer phenotype aids in theoptimization of ghost cell expansion, banking, cryopreservation, andthawing for use.

To prepare ghost cells, ghost cell trainers are treated with a specifichypotonic lytic procedure that extract all intracellular components, butallows plasma membrane, GPI anchored proteins, and lipid rafts to remainintact. Hypotonic lytic procedures for the preparation of ghost cellsare known in the art. Briefly, the ghost cell trainers are exposed to ahypotonic lytic solution containing cocktails to inhibit proteindegradation such as tyrosine, acid and alkaline phosphatases; serineproteases; trypsin, chymotrypsin, plasmin, and amino-peptidases;cysteine proteases; L-isozymes, and serine-threonine proteinphosphatases diluted in distilled water with a pH of 7.0. This hypotoniclytic solution will cause the initiation of caspase 3,8 andmitochondrial apoptotic cascade, as well as, create a large hole in theweaker side of the cell membrane allowing the entire intracellularcontents to leak out.

The protein of interest is an immunomodulatory molecule such as aco-stimulatory ligand, a co-stimulatory molecule, Fc receptor, or acytokine.

Fc receptors include for example CD64 or CD32.

Other proteins of interest include importin or GD2.

“Co-stimulatory ligand” refers to a molecule on an antigen presentingcell that specifically binds a cognate co-stimulatory molecule on aT-cell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation activation, differentiation and thelike. A co-stimulatory ligand can include but is not limited to CD7,B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL (CD137), OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM,CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin betareceptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Tollligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as but not limited to, CD-3ζ, CD27, CD28, 4-IBB, OX40, CD30, CD40,CD-40L, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1),CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds withCD83.

A “co-stimulatory molecule” refers to the cognate binding partner on aT-cell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the cell, such as, but notlimited to proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class 1 molecule, BTLA and Toll ligand receptor.Examples of costimulatory molecules include CD-3ζ, CD27, CD28, CD8,4-1BB (CD137),-1BBL OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 anda ligand that specifically binds with CD83 and the like. In anotherparticular embodiment, said signal transducing domain is aTNFR-associated Factor 2 (TRAF2) binding motifs, intracytoplasmic tailof costimulatory TNFR member family. Cytoplasmic tail of costimulatoryTNFR family member contains TRAF2 binding motifs consisting of the majorconserved motif (P/S/A)X(Q/E)E) or the minor motif (PXQXXD), wherein Xis any amino acid. TRAF proteins are recruited to the intracellulartails of many TNFRs in response to receptor trimerization.

By cytokine is meant any substance that is secreted by certain cells ofthe immune system and have an effect on other cells. Exemplary cytokinesinclude, but are not limited IL-1-like, IL-1α, IL-1β, IL-1RA, IL-18,Common g chain (CD132), IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, Common bchain (CD131), IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11, G-CSF, IL-12,LIF, OSM, IL-10-like, IL-10, IL-20, IL-21, IL-14, IL-16, IL-17, IFN-α,IFN-β, IFN-γ, CD154, LT-β, TNF-αTNF-β, APRIL, CD70, CD153, CD178, GITRL,LIGHT, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo,Flt-3L, SCF, M-CS, or any combinations thereof.

The protein of interest or MHC molecule may introduced by any methodsknow in the art. For example, expression vectors that encode the proteinof interest or MHC molecule can be introduced as one or more DNAmolecules or constructs. Optionally, there may be at least one markerthat will allow for selection of cells that contain the construct(s).

The constructs can be prepared in conventional ways, where the genes andregulatory regions may be isolated, as appropriate, ligated, cloned inan appropriate cloning host, analyzed by restriction or sequencing, orother convenient means. Particularly, using PCR, individual fragmentsincluding all or portions of a functional unit may be isolated, whereone or more mutations may be introduced using “primer repair”, ligation,in vitro mutagenesis, etc., as appropriate. The construct(s) oncecompleted and demonstrated to have the appropriate sequences may then beintroduced into the cell by any convenient means. The constructs may beintegrated and packaged into non-replicating, defective viral genomeslike Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus(HSV) or others, including retroviral vectors or lentiviral vectors, forinfection or transduction into cells. The constructs may include viralsequences for transfection, if desired. Alternatively, the construct maybe introduced by fusion, electroporation, biolistics, transfection,lipofection, or the like. The cells may be grown and expanded in culturebefore introduction of the construct(s), followed by the appropriatetreatment for introduction of the construct(s) and integration of theconstruct(s). The cells are then expanded and screened by virtue of amarker present in the construct. Various markers that may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can knock out an endogenous gene andreplace it (at the same locus or elsewhere) with the gene encoded for bythe construct using materials and methods known in the art forhomologous recombination. For homologous recombination, one may useeither .OMEGA. or O-vectors. See, for example, Thomas and Capecchi, Cell(1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; andJoyner, et al., Nature (1989) 338, 153-156.

Vectors containing useful elements such as bacterial or yeast origins ofreplication, selectable and/or amplifiable markers, promoter/enhancerelements for expression in prokaryotes or eukaryotes, etc. that may beused to prepare stocks of construct DNAs and for carrying outtransfections are well known in the art, and many are commerciallyavailable.

In addition, other methods known in the art may be used, such asself-inactivating transposase and transposon system, a drug selectionequipped DNA printed plasmid, or CRISPR.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the lactamase(penicillinase), lactose and tryptophan (trp) promoter systems. Inaddition to producing nucleic acid sequences of promoters and enhancerssynthetically, sequences may be produced using recombinant cloningand/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination could also be used todrive expression. Use of a T3, T7 or SP6 cytoplasmic expression systemis another possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages, and these may be used in the invention.

In certain embodiments of the invention, the 2A self cleaving peptidesare used to create multigene, or polycistronic, messages, and these maybe used in the invention.

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. “Restriction enzyme digestion” refers to catalyticcleavage of a nucleic acid molecule with an enzyme that functions onlyat specific locations in a nucleic acid molecule. Many of theserestriction enzymes are commercially available. Use of such enzymes iswidely understood by those of skill in the art. Frequently, a vector islinearized or fragmented using a restriction enzyme that cuts within theMCS to enable exogenous sequences to be ligated to the vector.“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments, which may or may not be contiguous with eachother. Techniques involving restriction enzymes and ligation reactionsare well known to those of skill in the art of recombinant technology.

Splicing sites, termination signals, origins of replication, andselectable markers may also be employed.

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™ 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Components of the present invention may be a viralvector that encodes one or peptides of interest. Non-limiting examplesof virus vectors that may be used to deliver a nucleic acid of thepresent invention are described below.

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the cells of the presentinvention as it has a high frequency of integration and it can infectnondividing cells, thus making it useful for delivery of genes intomammalian cells, for example, in tissue culture (Muzyczka, 1992) or invivo. AAV has a broad host range for infectivity (Tratschin et al.,1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,1988). Details concerning the generation and use of rAAV vectors aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference.

Retroviruses are useful as delivery vectors because of their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding the desired sequence) is inserted into the viral genome in theplace of certain viral sequences to produce a virus that is replicationdefective. In order to produce virions, a packaging cell line containingthe gag, pol, and env genes but without the LTR and packaging componentsis constructed (Mann et al., 1983). When a recombinant plasmidcontaining a cDNA, together with the retroviral LTR and packagingsequences is introduced into a special cell line (e.g., by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

Suitable methods for nucleic acid delivery for transfection ortransformation of cells are known to one of ordinary skill in the art.Such methods include, but are not limited to, direct delivery of DNA,RNA or mRNA such as by ex vivo transfection, by injection, and so forth.Through the application of techniques known in the art, cells may bestably or transiently transformed.

Methods of Immune Cell Expansion

Immune cells are activated in ex vivo by first isolating immune cellsfrom a subject sample. The sample is for example blood, bone marrow, ora tissue sample. For example, immune cells are isolated from peripheralblood mononuclear cells (PBMCs), bone marrow, or the spleen. Immunecells include B-cells, T cells, including a helper T cell (Th), acytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte, CTL,T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell) aregulatory T cell (Treg), a T follicular regulatory cell (TFR), NK cellsand NKT cells.

Immune cells are isolated by any methods known in the art. For example,immune cells are isolated by ficoll density centrifugation, flowcytometry, magnetic cell isolation and cell separation (MACS),RosetteSep, or antibody panning. One or more isolation techniques may beutilized in order to provide an immune cell population with sufficientpurity, viability, and yield.

The purity of the isolated immune cells is at least about 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, or more. The isolated immune cells are atleast about 70%, 75%, 80%, 85%, 90%, 95%, or more viable.

Optionally, after isolation the immune cells can be cryopreserved inin-house freeze media (FM) or commercially derived FM. FM may containthe following solutions: 40-70% Saline (PBS, HBSS, etc), 20-40% canine,feline, human, or equine serum, 1-10% Dimethyl sulfoxide (DMSO) and maybe filtered sterilized one to three times. The concentration range ofcells in the FM is between 5×10⁶ to 1×10⁸ cells/ml.

Immune cells are activated resulting in expansion by co-cultured withghost cells. Co-culturing also results in the immune cells having one ormore additional characteristics such as increased pro-inflammatorycytokine production, increased CD3zeta expression, increased ZAP70expression, upregulation of CD3, CD8; increased cell diameter andelongated morphology; increased homing and trafficking molecules likeCCR7, increased expression of perforin and cytolytic enzymes likegranzyme B.

Ghost cells will be added to the immune cell culture at a ratio rangingfrom (Ghost Cell: immune cell) 2:1 to 1:3.

This is a novel way of inducing activation. In contrast to the method ofthe invention, current methods of immune cell activation which utilizecesium irradiation to kill the tumor cell lines are more expensive andhave complex regulatory issues, making it difficult for therapeuticapplications. In addition, cesium irradiation the tumor cell linescontinue to function as highly viable artificial antigen presentingcells in culture for days after irradiation. These artificial antigenpresenting cells remain intact and have the capacity to proliferate,move, and secrete immunosuppressive cytokines. In contrast, theactivation though ghost cells according to the methods of the inventionis superior to previous methods as using ghost cells instead ofirradiated tumor cells line, alleviates the immunosuppressive effect ofcytokine secretion; tumor cell line clustering to inhibit T-cellmediated lysis; T-cell tolerance to tumor antigens, and possible tumorproliferation and viability. In addition the methods of the inventionwimprove synaptic formation and strength to increase immune cellexpansion.

In some embodiments, the immune cells are activated in in RPMI, 1-100%100×glutamax, 1-100% FBS) at a concentration of 1.5 to 2×10⁶ cells/ml.

In some embodiments, the cells are co-cultured in a cell culture mediumcontaining a cytokine. The cytokine is, for example, IL-1-like, IL-1α,IL-1β, IL-1RA, IL-18, Common g chain (CD132), IL-2, IL-4, IL-7, IL-9,IL-13, IL-15, Common b chain (CD131), IL-3, IL-5, GM-CSF, IL-6-like,IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL-21,IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD154, LT-β, TNF-αTNF-β,4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL,TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF,αCD40, or any combinations thereof. Toll-like receptor agonist may alsobe used. Preferably the cytokine is IL-2, and/or IL-21.

The cytokine is added to the cultures 2 to 3 times per week at aconcentration of and, respectively, 10 to 100 U/mL or IL-2) and/or 25 to75 ug/mL of IL-21. In some embodiments IL-2 is not added for the first 7to 10 days of culture.

Immune cells will be re-stimulated with ghost cells every 4 to 8 daysuntil a clinically sufficient number of cells has been reached for eachsubject. The clinically significant numbers is based on body surfacearea (BSA) calculated from the weight of the subject.

Immune cells will be re-stimulated with ghost cells every 4 to 8 daysuntil a clinically sufficient number of cells has been reached for eachsubject. The clinically significant numbers is based on body surfacearea (BSA) calculated from the weight of the subject.

The expanded oligoclonal immune cells may be cryopreserved in in-housefreeze media (FM) or commercially derived FM prior to infusion into asubject. FM may contain the following solutions: 40-70% Saline (PBS,HBSS, etc), 20-40% canine, feline, human, or equine serum, 1-10%Dimethyl sulfoxide (DMSO) and may be filtered sterilized one to threetimes. The concentration range of cells in the FM is between 5×10⁶ to1×10⁸ cells/ml.

Therapeutic Methods

The reagents according to the invention can be used for treating cancer,infection or other immunological disorders such as graft vs host diseaseor autoimmune disorders in a subject in need thereof. In anotherembodiment, reagents according to the invention can be used in themanufacture of a medicament for treatment of a cancer, infection orother immunological disorders such as graft vs host disease orautoimmune disorders in a subject in need thereof. The present inventionrelies on methods for treating subjects in need thereof by administeringto the subject a composition containing the oligoclonal immune cellsexpanded by the methods of the invention The subject is a mammal.Mammals include, but are not limited to, humans, farm animals, sportanimals (e.g., horses), and companion animals (e.g., dog or cats).

Treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said subject or from a Leucocyte Antigen (LA) compatible donor. Byallogeneic is meant that the cells or population of cells used fortreating subjects are not originating from the subject but from amatching (i.e., hostocompatiable) donor.

Treatment is efficacious if the treatment leads to clinical benefit suchas, a decrease in size, prevalence, or metastatic potential of the tumorin the subject. When treatment is applied prophylactically,“efficacious” means that the treatment retards or prevents tumors fromforming or prevents or alleviates a symptom of clinical symptom of thetumor. Efficaciousness is determined in association with any knownmethod for diagnosing or treating the particular tumor type.

Cells and methods of producing them that can be used with the disclosedmethods are described herein. Treatment can be used to treat patientsdiagnosed with cancer, autoimmune disorders or Graft versus Host Disease(GvHD). Cancers that may be treated include tumors that are notvascularized, or not yet substantially vascularized, as well asvascularized tumors. The cancers may comprise nonsolid tumors such ashematological tumors, for example, leukemias and lymphomas. The leukemiais acute myeloid leukemia (AML), chronic myeloid leukemia (CIVIL), acutelymphocytic leukemia (ALL) or chronic lymphocytic leukemic (CLL).Alternatively, the tumor is a solid tumor. Solid tumors include, but arenot limited to, carcinoma; such as transitional cell carcinoma, mammarycarcinoma, or carcinoma of the gastrointestinal system; sarcoma such asosteosarcoma, hemangiosarcoma or sarcoma of the gastrointestinal system;blastoma such as neuroblastoma or glioblastoma; or melanoma.

Treatment may be in combination with one or more therapies againstcancer selected from the group of antibodies therapy, chemotherapy,cytokines therapy, dendritic cell therapy, stem cell therapy, genetherapy, hormone therapy, laser light therapy and radiation therapyprior to or after treatment according to the methods of the invention.

For example the subject has received HSCT, targeted or whole bodyirradiation or high dose Cytoxan therapy, prior to the administration ofthe cell population. For example, cells are administered 7-14 days posthigh dose Cytoxan therapy. For example, cells are administered 24-72hours post targeted or whole body radiation.

Therapeutic Administration

The invention includes administering to a subject therapeuticcomposition comprising the expanded oligoclonal immune cells produced bythe methods of the invention.

Effective doses vary, as recognized by those skilled in the art,depending on route of administration, excipient usage, andcoadministration with other therapeutic treatments including use ofother anti-proliferative agents or therapeutic agents for treating,preventing or alleviating a symptom of cancer, infection or otherimmunological disorders such as graft vs host disease or autoimmunedisorders. A therapeutic regimen is carried out by identifying a mammal,e.g., a suffering from a cancer, infection or other immunologicaldisorders such as graft vs host disease or autoimmune disorders usingstandard methods.

Compositions containing the appropriate expanded oligoclonal immunecells are administered to an individual in a regimen determined asappropriate by a person skilled in the art. For example, the compositionmay be given multiple times at an appropriate interval and dosage.

The cells may be administered as desired. Depending upon the responsedesired, the manner of administration, the life of the cells, the numberof cells present, various protocols may be employed. Depending upon thenature of the cells, the cells may be introduced into a host organism,e.g. a mammal, in a wide variety of ways. The cells may be introduced atthe site of the tumor, in specific embodiments, although in alternativeembodiments the cells hone to the cancer or are modified to hone to thecancer. The number of cells that are employed will depend upon a numberof circumstances, the purpose for the introduction, the lifetime of thecells, the protocol to be used, for example, the number ofadministrations, the ability of the cells to multiply, the stability ofthe recombinant construct, and the like. The cells may be applied as adispersion, generally being injected at or near the site of interest.The cells may be in a physiologically-acceptable medium.

In some embodiments, the cells are encapsulated to inhibit immunerecognition and placed at the site of the tumor.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includinginjection, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermaly, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous or intralymphaticinjection, or intraperitoneally. In one embodiment, the cellcompositions of the present invention are preferably administered byintravenous injection. Additionally, compositions are administered byimplanting (either directly into an organ or subcutaneously) a solid orresorbable matrix which slowly releases the composition into adjacentand surrounding tissues of the subject.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. An appropriate does is for example 25-300 ml,25-200 ml, 25-100 ml of a composition containing approximately 10⁵-10¹⁰cell/m², preferably about 1×10⁷ to 5×10⁹ cell/m², preferably the dose is5×10⁸ cell/m².

The cells or population of cells can be administrated in one or moredoses. In another embodiment, said effective amount of cells areadministrated as a single dose. In another embodiment, said effectiveamount of cells are administrated as more than one dose over a periodtime. Doses may be administered once, or more than once. In someembodiments, it is preferred that the therapeutic composition isadministered once every 7 to 10 days, The predetermined duration of timemay be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, or up to 1 year.

Timing of administration is within the judgment of managing physician orveterinarian and depends on the clinical condition of the subject. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of a given cell type for a particular disease or conditionswithin the skill of the art. An effective amount means an amount whichprovides a therapeutic or prophylactic benefit. The dosage administratedwill be dependent upon the age, health and weight of the recipient, kindof concurrent treatment, if any, frequency of treatment and the natureof the effect desired.

The composition of expanded oligoclonal immune cells prior toadministration to the subject must have sufficient viability. Theviability of the fused cells at the time of administration is at least50%, at least 60%, at least 70%, at least 80% or greater.

Prior to administration, the population of expanded oligoclonal immunecells are free of components used during the production, e.g., cellculture components and substantially free of mycoplasm, endotoxin, andmicrobial contamination. Preferably, the population of fused cells hasless than 10, 5, 3, 2, or 1 CFU/swab. Most preferably the population ofexpanded oligoclonal immune cells has 0 CFU/swab. For example, theresults of the sterility testing is “negative” or “no growth”. Theendotoxin level in the population of tumor cells is less than 20 EU/mL,less than 10 EU/mL or less than 5 EU/mL. The result of the myoplasmtesting is “negative”.

For non-food animal use all final cell products must conform to therequirements imposed by the USDA under 9CFR Part103.3.

For human use, all final cell product must conform with rigidrequirements imposed by the Federal Drug Administration (FDA). The FDArequires that all final cell products must minimize “extraneous”proteins known to be capable of producing allergenic effects in humansubjects as well as minimize contamination risks. Moreover, the FDAexpects a minimum cell viability of 70%, and any process shouldconsistently exceed this minimum requirement.

In some embodiments, it is preferred that the therapeutic compoundsdescribed herein are administered in combination with anothertherapeutic agent, such as a chemotherapeutic agent, radiation therapy,or an anti-mitotic agent. In some aspects, the anti-mitotic agent isadministered prior to administration of the present therapeuticcompound, in order to induce additional chromosomal instability toincrease the efficacy of the present invention to targeting cancercells. Examples of anti-mitotic agents include taxanes (i.e.,paclitaxel, docetaxel), and vinca alkaloids (i.e., vinblastine,vincristine, vindesine, vinorelbine).

Identification of T-Cell Receptor Sequences

In another object of the invention T-cell receptor (TCR) sequence of theexpanded T-cells of the invention are identified. Alternatively, thenative TCR sequences are mutated and the TCR tumor antigen pairings areidentified. Methods of mutating TCR sequences are known in the art andinclude SHM or AID.

Native TCR sequences or mutated TCR sequence are used to create achimeric antigen receptor cell. (CAR), that are then used to treatdiseases and disorders as described in the method above.

The CAR according to the invention generally comprises at least onetransmembrane polypeptide comprising at least one extracellularligand-binding domain and; one transmembrane polypeptide comprising atleast one intracellular signaling domain; such that the polypeptidesassemble together to form a Chimeric Antigen Receptor.

The term “extracellular ligand-binding domain” as used herein is definedas an polypeptide that is capable of binding a ligand. Preferably, thedomain will be capable of interacting with a cell surface molecule. Forexample, the extracellular ligand-binding domain may be chosen torecognize a ligand that acts as a cell surface marker on target cellsassociated with a particular disease state. Most preferably, theextracellular domain is the variable region of a T-cell receptor.

In particular, the extracellular ligand-binding domain comprises thevariable region of a T-cell receptor specific for a tumor associatedantigen or a self antigen. Preferably, the T-cell receptor, the tumorassociated antigen or self antigen has been identified from a T-cellobtained from a subject. For example, the T-cell receptor is identifiedfrom a tumor infiltrating lymphocyte, a lymphocyte from an autoimmunesite or a lymphocyte from a graft tissue.

In a preferred embodiment said transmembrane domain further comprises astalk region between said extracellular ligand-binding domain and saidtransmembrane domain. The term “stalk region” used herein generallymeans any oligo- or polypeptide that functions to link the transmembranedomain to the extracellular ligand-binding domain. In particular, stalkregions are used to provide more flexibility and accessibility for theextracellular ligand-binding domain. A stalk region may comprise up to300 amino acids, preferably 10 to 100 amino acids more preferably 25 to50 amino acids and most preferably 3 to 15 amino acids. Stalk region maybe derived from all or part of naturally occurring molecules, such asfrom all or part of the extracellular region of CD8, CD4 or CD28, orfrom all or part of an antibody constant region. Alternatively the stalkregion may be a synthetic sequence that corresponds to a naturallyoccurring stalk sequence, or may be an entirely synthetic stalksequence. In a preferred embodiment said stalk region is a part of humanCD8 alpha chain.

The signal transducing domain or intracellular signaling domain of theCAR of the invention is responsible for intracellular signalingfollowing the binding of extracellular ligand binding domain to thetarget resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the CAR is expressed. For example, the effectorfunction of a T cell can be a cytolytic activity or helper activityincluding the secretion of cytokines. Thus, the term “signal transducingdomain” refers to the portion of a protein which transduces the effectorsignal function signal and directs the cell to perform a specializedfunction.

Signal transduction domain comprises two distinct classes of cytoplasmicsignaling sequence, those that initiate antigen-dependent primaryactivation, and those that act in an antigen-independent manner toprovide a secondary or co-stimulatory signal. Primary cytoplasmicsignaling sequence can comprise signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are welldefined signaling motifs found in the intracytoplasmic tail of a varietyof receptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCR zeta, FcR gamma, FcR beta, FcRepsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b andCD66d. In a preferred embodiment, the signaling transducing domain ofthe CAR can comprise the CD3 zeta signaling domain, or theintracytoplasmic domain of the Fc epsilon RI beta or gamma chains. Inanother preferred embodiment, the signaling is provided by CD3 zetatogether with co-stimulation provided by CD28 and a tumor necrosisfactor receptor (TNFr), such as 4-1BB or OX40), for example.

In particular embodiment the intracellular signaling domain of the CARof the present invention comprises a co-stimulatory signal molecule. Insome embodiments the intracellular signaling domain contains 2, 3, 4 ormore co-stimulatory molecules in tandem. A co-stimulatory molecule is acell surface molecule other than an antigen receptor or their ligandsthat is required for an efficient immune response.

The distinguishing features of appropriate transmembrane polypeptidescomprise the ability to be expressed at the surface of an immune cell,in particular lymphocyte cells or Natural killer (NK) cells, and tointeract together for directing cellular response of immune cell againsta predefined target cell. The different transmembrane polypeptides ofthe CAR of the present invention comprising an extracellularligand-biding domain and/or a signal transducing domain interacttogether to take part in signal transduction following the binding witha target ligand and induce an immune response. The transmembrane domaincan be derived either from a natural or from a synthetic source. Thetransmembrane domain can be derived from any membrane-bound ortransmembrane protein.

The term “a part of” used herein refers to any subset of the molecule,that is a shorter peptide. Alternatively, amino acid sequence functionalvariants of the polypeptide can be prepared by mutations in the DNAwhich encodes the polypeptide. Such variants or functional variantsinclude, for example, deletions from, or insertions or substitutions of,residues within the amino acid sequence. Any combination of deletion,insertion, and substitution may also be made to arrive at the finalconstruct, provided that the final construct possesses the desiredactivity, especially to exhibit a specific anti-target cellular immuneactivity. The functionality of the CAR of the invention within a hostcell is detectable in an assay suitable for demonstrating the signalingpotential of said CAR upon binding of a particular target. Such assaysare available to the skilled person in the art. For example, this assayallows the detection of a signaling pathway, triggered upon binding ofthe target, such as an assay involving measurement of the increase ofcalcium ion release, intracellular tyrosine phosphorylation, inositolphosphate turnover, or interleukin (IL) 2, interferon gamma., GM-CSF,IL-3, IL-4 production thus effected.

Embodiments of the invention include cells that express a CAR (i.e,CARTS). The cell may be of any kind, including an immune cell capable ofexpressing the CAR for cancer therapy or a cell, such as a bacterialcell, that harbors an expression vector that encodes the CAR. As usedherein, the terms “cell,” “cell line,” and “cell culture” may be usedinterchangeably. All of these terms also include their progeny, which isany and all subsequent generations. It is understood that all progenymay not be identical due to deliberate or inadvertent mutations. In thecontext of expressing a heterologous nucleic acid sequence, “host cell”refers to a eukaryotic cell that is capable of replicating a vectorand/or expressing a heterologous gene encoded by a vector. A host cellcan, and has been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid. In embodiments of the invention, a host cell is a T cell,including a helper T cell (Th), a cytotoxic T cell (also known as TC,Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+T-cells or killer T cell) a regulatory T cell (Treg), a T follicularregulatory cell (TFR), NK cells and NKT cells are also encompassed inthe invention.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

The cells can be autologous cells, syngeneic cells, allogenic cells andeven in some cases, xenogeneic cells.

The invention further includes CARTS that are modified to secrete one ormore polypeptides. The polypeptide can be for example an antibody orcytokine.

Armed CARTS have the advantage of simultaneously secreting a polypeptideat the targeted site, e.g. tumor site, graft site or autoimmune site.

Expression vectors that encode the CARs can be introduced as one or moreDNA molecules or constructs, where there may be at least one marker thatwill allow for selection of host cells that contain the construct(s).

The constructs can be prepared in conventional ways, where the genes andregulatory regions may be isolated, as appropriate, ligated, cloned inan appropriate cloning host, analyzed by restriction or sequencing, orother convenient means.

Identification of Immune Cell Epitopes

Another object of the invention is the identification of immune cell,e.g. T-cell epitopes of an antigen, which method allows for simultaneousand rapid examination of a large number of peptide sequences, for theircapability of binding to specific MHC molecules.

Specifically, the invention provides method of identifying and/ordetecting tumor specific neoantigens that are useful in inducing a tumorspecific immune response in a subject.

The molecules which transport and present peptides on the cell surfaceare referred to as proteins of the major histocompatibility complex(MHC). The MHC proteins present mainly peptides, which are processedfrom external antigen sources, i.e. outside of the cells, to T-helper(Th) cells. The peptides attach themselves to the molecules of MHC classI by competitive affinity binding within the endoplasmic reticulum,before they are presented on the cell surface. The affinity of anindividual peptide is directly linked to its amino acid sequence and thepresence of specific binding motifs in defined positions within theamino acid sequence. If the sequence of such a peptide is known, it ispossible, for example, to manipulate the immune system against diseasedcells using, for example, peptide vaccines. Using computer algorithms,it is possible to predict potential affinity.

The present invention is based, on the identification of certainmutations (e.g., the variants or alleles that are present in cancer ordiseased cells). In particular, these mutations are present in thegenome of cancer or diseased cells of a subject having cancer or anotherimmunodysfunction but not in normal tissue from the subject.

In particular, the invention provides a method of vaccinating ortreating a subject by identifying a plurality of disease specificmutations in the genome of a subject. Mutant peptides and polypeptideshaving the identified mutations and that binds to a class I HLA proteinare selected. Optionally, these peptide and polypeptides binds to aclass I HLA proteins with a greater affinity than the wild-type peptideand/or are capable of activating anti-tumor CD8 T-cells. These peptidesare administered to the subject as a vaccine. Alternatively, thepeptides are used to pulse immune cells or CAR cells which are thenadministered to the subject.

The invention further includes isolated peptides that comprise thedisease specific mutations identified by the methods of the invention.These peptides and polypeptides are referred to herein as “neoantigenicpeptides” or “neoantigenic polypeptides”. The term “peptide” is usedinterchangeably with “mutant peptide” and “neoantigenic peptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids.Similarly, the term “polypeptide” is used interchangeably with “mutantpolypeptide” and “neoantigenic polypeptide” in the present specificationto designate a series of residues, typically L-amino acids, connectedone to the other, typically by peptide bonds between the α-amino andcarboxyl groups of adjacent amino acids. The polypeptides or peptidescan be a variety of lengths, either in their neutral (uncharged) formsor in forms which are salts, and either free of modifications such asglycosylation, side chain oxidation, or phosphorylation or containingthese modifications, subject to the condition that the modification notdestroy the biological activity of the polypeptides as herein described.

In certain embodiments the size of the at least one neoantigenic peptidemolecule may comprise, but is not limited to, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 60, about 70, about 80, about 90, about 100, about 110,about 120 or greater amino molecule residues, and any range derivabletherein. In specific embodiments the neoantigenic peptide molecules areequal to or less than 50 amino acids.

In some embodiments the particular neoantigenic peptides andpolypeptides of the invention are: for WIC Class I 13 residues or lessin length and usually consist of between about 8 and about 11 residues,particularly 9 or 10 residues; for MHC Class II, 15-24 residues.

The neoantigenic peptides and polypeptides bind an HLA protein. In someaspect the neoantigenic peptides and polypeptides binds an HLA proteinwith greater affinity than a wild-type peptide. The neoantigenic peptideor polypeptide has an IC50 of at least less than 5000 nM, at least lessthan 500 nM, at least less than 250 nM, at least less than 200 nM, atleast less than 150 nM, at least less than 100 nM, at least less than 50nM or less.

The neoantigenic peptides and polypeptides do not induce an autoimmuneresponse and/or invoke immunological tolerance when administered to asubject.

The present invention is directed to an immunogenic composition, e.g., avaccine composition capable of raising a specific T-cell response. Thevaccine composition comprises mutant peptides and mutant polypeptidescorresponding to disease specific neoantigens identified by the methodsdescribed herein.

A person skilled in the art will be able to select preferred peptides,polypeptide or combination of thereof by testing, for example, thegeneration of T-cells in vitro as well as their efficiency and overallpresence, the proliferation, affinity and expansion of certain T-cellsfor certain peptides, and the functionality of the T-cells, e.g. byanalyzing the IFN-γ production or tumor killing by T-cells. Usually, themost efficient peptides are then combined as a vaccine.

Definitions

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

As used herein, the T lymphocyte can be any T lymphocyte, such as acultured T lymphocyte, e.g., a primary T lymphocyte, or a T lymphocytefrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a Tlymphocyte obtained from a mammal. If obtained from a mammal, the Tlymphocyte can be obtained from numerous sources, including but notlimited to blood, bone marrow, lymph node, the thymus, or other tissuesor fluids. T lymphocytes can also be enriched for or purified.Preferably, the T lymphocyte is a human T lymphocyte. More preferably,the T lymphocyte is a T lymphocyte isolated from a human. The Tlymphocyte can be any type of T lymphocyte and can be of anydevelopmental stage, including but not limited to, CD4⁺/CD8.⁺ doublepositive T lymphocytes, CD4⁺ helper T lymphocytes, e.g., Th₁ and Th₂cells, CD8⁺ T lymphocytes (e.g., cytotoxic T lymphocytes), peripheralblood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs),tumor infiltrating lymphocytes (TILs), memory T cells, naive Tlymphocytes, and the like. Preferably, the T lymphocyte is a TIL or aPBMC.

As used herein, “NK cells” means cytotoxic effector cells with thecapacity to lyse tissue culture cells without participation of anantibody and without in vitro or in vivo sensitization. NK cells mayalso be characterized by the presence of cell surface receptors orproteins that distinguish NK cells from other lymphoid cells, and cellsof the erythroid or myeloid lineages, for example, see, Bradshaw et al.,Handbook of Cell Signaling (2003).

As used herein, “cells capable of differentiation into NK cells” refersto HSCs that differentiate into NK cells, when Jagged2, Flt3L, IL-7, andSCF are expressed in co-cultured cells or added to the growth media.Optionally, IL-2 may be supplied. Cells capable of differentiation intoNK cells may be genetically modified either in vivo or in vitro, forexample, reporter constructs may be introduced, or therapeutic geneproducts may by introduced or alternatively regulated.

The term “immune cells” refers to cells that specifically recognize anantigen present, for example on a neoplastic or tumor cell. For thepurposes of this invention, immune effector cells include, but are notlimited to, B cells; monocytes; macrophages; NK cells; and T cells suchas cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,and CTLs from tumor, inflammatory sites or other infiltrates.“T-lymphocytes” denotes lymphocytes that are phenotypically CD3+,typically detected using an anti-CD3 monoclonal antibody in combinationwith a suitable labeling technique. The T-lymphocytes of this inventionare also generally positive for CD4, CD8, or both. The term “naïve”immune effector cells refers to immune effector cells that have notencountered antigen and is intended to by synonymous with unprimed andvirgin. “Educated” refers to immune effector cells that have interactedwith an antigen such that they differentiate into an antigen-specificcell.

The terms “antigen presenting cells” or “APCs” includes both intact,whole cells as well as other molecules which are capable of inducing thepresentation of one or more antigens, preferably with class I MHCmolecules. Examples of suitable APCs are discussed in detail below andinclude, but are not limited to, whole cells such as macrophages,dendritic cells, B cells; purified MHC class I molecules complexed toβ2-microglobulin; and foster antigen presenting cells.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. As used herein,“ameliorated” or “treatment” refers to a symptom which is approaches anormalized value (for example a value obtained in a healthy patient orindividual), e.g., is less than 50% different from a normalized value,preferably is less than about 25% different from a normalized value,more preferably, is less than 10% different from a normalized value, andstill more preferably, is not significantly different from a normalizedvalue as determined using routine statistical tests.

Thus, treating may include suppressing, inhibiting, preventing,treating, or a combination thereof. Treating refers inter alia toincreasing time to sustained progression, expediting remission, inducingremission, augmenting remission, speeding recovery, increasing efficacyof or decreasing resistance to alternative therapeutics, or acombination thereof. “Suppressing” or “inhibiting”, refers inter alia todelaying the onset of symptoms, preventing relapse to a disease,decreasing the number or frequency of relapse episodes, increasinglatency between symptomatic episodes, reducing the severity of symptoms,reducing the severity of an acute episode, reducing the number ofsymptoms, reducing the incidence of disease-related symptoms, reducingthe latency of symptoms, ameliorating symptoms, reducing secondarysymptoms, reducing secondary infections, prolonging patient survival, ora combination thereof. The symptoms are primary, while in anotherembodiment, symptoms are secondary. “Primary” refers to a symptom thatis a direct result of the proliferative disorder, while, secondaryrefers to a symptom that is derived from or consequent to a primarycause. Symptoms may be any manifestation of a disease or pathologicalcondition.

The “treatment of cancer or tumor cells”, refers one or more of thefollowing effects: (1) inhibition of tumor growth, including, (i)slowing down and (ii) complete growth arrest; (2) reduction in thenumber of tumor cells; (3) maintaining tumor size; (4) reduction intumor size; (5) inhibition, including (i) reduction, (ii) slowing downor (iii) complete prevention, of tumor cell infiltration into peripheralorgans; (6) inhibition, including (i) reduction, (ii) slowing down or(iii) complete prevention, of metastasis; (7) enhancement of anti-tumorimmune response, which may result in (i) maintaining tumor size, (ii)reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing,slowing or preventing invasion and/or (8) relief, to some extent, of theseverity or number of one or more symptoms associated with the disorder.

As used herein, “an ameliorated symptom” or “treated symptom” refers toa symptom which approaches a normalized value, e.g., is less than 50%different from a normalized value, preferably is less than about 25%different from a normalized value, more preferably, is less than 10%different from a normalized value, and still more preferably, is notsignificantly different from a normalized value as determined usingroutine statistical tests.

The terms “patient” or “individual” are used interchangeably herein, andrefers to a mammalian subject to be treated.

By the term “modulate,” it is meant that any of the mentionedactivities, are, e.g., increased, enhanced, increased, augmented,agonized (acts as an agonist), promoted, decreased, reduced, suppressedblocked, or antagonized (acts as an antagonist). Modulation can increaseactivity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold,etc., over baseline values. Modulation can also decrease its activitybelow baseline values.

As used herein, the term “administering to a cell” (e.g., an expressionvector, nucleic acid, a delivery vehicle, agent, and the like) refers totransducing, transfecting, microinjecting, electroporating, or shooting,the cell with the molecule. In some aspects, molecules are introducedinto a target cell by contacting the target cell with a delivery cell(e.g., by cell fusion or by lysing the delivery cell when it is inproximity to the target cell).

Thus, the term “cytokine” refers to any of the numerous factors thatexert a variety of effects on cells, for example, inducing growth orproliferation. Non-limiting examples of cytokines include, IL-2, stemcell factor (SCF), IL-3, IL-6, IL-7, IL-12, IL-15, G-CSF, GM-CSF, IL-1α, IL-1 β, MIP-1 α, LIF, c-kit ligand, TPO, and flt3 ligand. Cytokinesare commercially available from several vendors such as, for example,Genzyme Corp. (Framingham, Mass.), Genentech (South San Francisco,Calif.), Amgen (Thousand Oaks, Calif.) and Immunex (Seattle, Wash.). Itis intended, although not always explicitly stated, that moleculeshaving similar biological activity as wild-type or purified cytokines(e.g., recombinantly produced cytokines) are intended to be used withinthe spirit and scope of the invention and therefore are substitutes forwild-type or purified cytokines.

“Costimulatory molecules” are involved in the interaction betweenreceptor-ligand pairs expressed on the surface of antigen presentingcells and T cells. One exemplary receptor-ligand pair is the B7co-stimulatory molecules on the surface of DCs and its counter-receptorCD28 or CTLA-4 on T cells. (See Freeman et al. (1993) Science262:909-911; Young et al. (1992) J. Clin. Invest 90: 229; Nabavi et al.Nature 360:266)). Other important costimulatory molecules include, forexample, CD40, CD54, CD80, and CD86.

The term “culturing” refers to the in vitro propagation of cells ororganisms on or in media of various kinds, it is understood that thedescendants of a cell grown in culture may not be completely identical(i.e., morphologically, genetically, or phenotypically) to the parentcell. By “expanded” is meant any proliferation or division of cells.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

An “isolated” population of cells is “substantially free” of cells andmaterials with which it is associated in nature.

By “substantially free” or “substantially pure” is meant at least 50% ofthe population are the desired cell type, preferably at least 70%, morepreferably at least 80%, and even more preferably at least 90%.

An “enriched” population of cells is at least 5% of the desired celltype. Preferably, the enriched population contains at least 10%, morepreferably at least 20%, and most preferably at least 25% of the desiredcell type.

The term “autogeneic”, or “autologous”, as used herein, indicates theorigin of a cell. Thus, a cell being administered to an individual (the“recipient”) is autogeneic if the cell was derived from that individual(the “donor”) or a genetically identical individual (i.e., an identicaltwin of the individual). An autogeneic cell can also be a progeny of anautogeneic cell. The term also indicates that cells of different celltypes are derived from the same donor or genetically identical donors.

Similarly, the term “allogeneic”, as used herein, indicates the originof a cell. Thus, a cell being administered to an individual (the“recipient”) is allogeneic if the cell was derived from an individualnot genetically identical to the recipient. In particular, the termrelates to non-identity in expressed MEW molecules. An allogeneic cellcan also be a progeny of an allogeneic cell. The term also indicatesthat cells of different cell types are derived from geneticallynonidentical donors, or if they are progeny of cells derived fromgenetically non-identical donors.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and companion animals.

As used herein, “genetic modification” refers to any addition, deletionor disruption to a cell's endogenous nucleotides.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors and the like. In aspects where gene transfer is mediated by aretroviral vector, a vector construct refers to the polynucleotidecomprising the retroviral genome or part thereof, and a therapeuticgene.

As used herein, the terms “retroviral mediated gene transfer” or“retroviral transduction” carries the same meaning and refers to theprocess by which a gene or a nucleic acid sequence is stably transferredinto the host cell by virtue of the virus entering the cell andintegrating its genome into the host cell genome. The virus can enterthe host cell via its normal mechanism of infection or be modified suchthat it binds to a different host cell surface receptor or ligand toenter the cell.

Retroviruses carry their genetic information in the form of RNA.However, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form that integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas a adenovirus (Ad) or adeno-associated virus (AAV), a vector constructrefers to the polynucleotide comprising the viral genome or partthereof, and a therapeutic gene. Adenoviruses (Ads) are a relativelywell characterized, homogenous group of viruses, including over 50serotypes. (See, e.g., WO 95/27071). Ads are easy to grow and do notintegrate into the host cell genome. Recombinant Ad-derived vectors,particularly those that reduce the potential for recombination andgeneration of wild-type virus, have also been constructed. (See, WO95/00655; WO 95/11984). Wild-type AAV has high infectivity andspecificity integrating into the host cells genome. (See Hermonat andMuzyczka (1984) PNAS USA 81:6466-6470; Lebkowski et al., (1988) Mol CellBiol 8:3988-3996).

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression. Examples ofsuitable vectors are viruses, such as baculovirus and retrovirus,bacteriophage, cosmid, plasmid, fungal vectors and other recombinationvehicles typically used in the art which have been described forexpression in a variety of eucaryotie and prokaryotic hosts, and may beused for gene therapy as well as for simple protein expression.

Among these are several non-viral vectors, including DNA/liposomecomplexes, and targeted viral protein DNA complexes. Liposomes that alsocomprise a targeting antibody or fragment thereof can be used in themethods of this invention. This invention also provides the targetingcomplexes for use in the methods disclosed herein.

Polynucleotides are inserted into vector genomes using methods wellknown in the art. For example, insert and vector DNA can be contacted,under suitable conditions, with a restriction enzyme to createcomplementary ends on each molecule that can pair with each other and bejoined together with a ligase. Alternatively, synthetic nucleic acidlinkers can be ligated to the termini of restricted polynucleotide.These synthetic linkers contain nucleic acid sequences that correspondto a particular restriction site in the vector DNA. Additionally, anoligonucleotide containing a termination codon and an appropriaterestriction site can be ligated for insertion into a vector containing,for example, some or all of the following: a selectable marker gene,such as the neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and ColEI for properepisomal replication; versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Other means are well known and available in the art.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeukaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG (Sambrook et al. (1989), supra). Similarly, a eukaryotic expressionvector includes a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Such vectors can beobtained commercially or assembled by the sequences described in methodswell known in the art, for example, the methods described above forconstructing vectors in general.

The terms “major histocompatibility complex” or “MHC” refers to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to immune effector cells such as T cells. Inhumans, the MEW complex is also known as the HLA complex. Indogs the MEWcomplex is also known as the DLA complex. The proteins encoded by theMEW complex are known as “MHC molecules” In humans MHC molecules areclassified into class I and class II MEW molecules. Human Class I MEWmolecules include membrane heterodimeric proteins made up of an a chainencoded in the MEW associated noncovalently with β2-microglobulin. HumanClass I MEW molecules are expressed by nearly all nucleated cells andhave been shown to function in antigen presentation to CD8+ T cells.Human Class I molecules include HLA-A, -B, and -C in humans. Human ClassII MEW molecules also include membrane heterodimeric proteins consistingof noncovalently associated and J3 chains. Human Class II MHCs are knownto function in CD4+ T cells and, in humans, include HLA-DP, -DQ, and DR.In dogs, the MEW molecules are classified into class I, class II andclass III molecules. Canine Class I MHC molecules are expressed bynearly all nucleated cells and have been shown to function in antigenpresentation to T cells. Canine Class II molecules are expressed onantigen-presenting cells. Canine Class III molecules are not relevant toantigen presentation but can be important in other aspects of the immunesystem such as complement activation.

The term “MHC restriction” refers to a characteristic of T cells thatpermits them to recognize antigen only after it is processed and theresulting antigenic peptides are displayed in association with either aclass I or class II MEW molecule. Methods of identifying and comparingMEW are well known in the art and are described in Allen M. et al.(1994) Human Imm. 40:25-32; Santamaria P. et al. (1993) Human Imm.37:39-50; and Hurley C. K. et al. (1997) Tissue Antigens 50:401-415.

The term “sequence motif” refers to a pattern present in a group of 15molecules (e.g., amino acids or nucleotides). For instance, in oneembodiment, the present invention provides for identification of asequence motif among peptides present in an antigen. In this embodiment,a typical pattern may be identified by characteristic amino acidresidues, such as hydrophobic, hydrophilic, basic, acidic, and the like.

The term “peptide” is used in its broadest sense to refer to a compoundof two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc.

As used herein the term “amino acid” refers to either natural and/or 25unnatural or synthetic amino acids, including glycine and both the D orL optical isomers, and amino acid analogs and peptidomimetics. A peptideof three or more amino acids is commonly called an oligopeptide if thepeptide chain is short. If the peptide chain is long, the peptide iscommonly called a polypeptide or a protein.

As used herein, “solid phase support” is used as an example of a“carrier” and is not limited to a specific type of support. Rather alarge number of supports are available and are known to one of ordinaryskill in the art. Solid phase supports include silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels. A suitable solid phase support may be selected on the basis ofdesired end use and suitability for various synthetic protocols. Forexample, for peptide synthesis, solid phase support may refer to resinssuch as polystyrene (e.g., PAM-resin obtained from Bachem Inc.,Peninsula Laboratories, etc.), POLYHIPE® resin (obtained from Aminotech,Canada), polyamide resin (obtained from Peninsula Laboratories),polystyrene resin grafted with polyethylene glycol (TentaGel®, RappPolymere, Tubingen, Germany) or polydimethylacrylamide resin (obtainedfrom MilligenlBiosearch, Calif.). In a preferred embodiment for peptidesynthesis, solid phase support refers to polydimethylacrylamide resin.

“Host cell” or “recipient cell” is intended to include any individualcell or cell culture which can be or have been recipients for vectors orthe incorporation of exogenous nucleic acid molecules, polynucleotidesand/or proteins. It also is intended to include progeny of a singlecell, and the progeny may not necessarily be completely identical (inmorphology or in genomic or total DNA complement) to the original parentcell due to natural, accidental, or deliberate mutation. The cells maybe prokaryotic or eukaryotic, and include but are not limited tobacterial cells, yeast cells, animal cells, and mammalian cells, e.g.,murine, rat, simian or human.

An “antibody” is an immunoglobulin molecule capable of binding anantigen. As used herein, the term encompasses not only intactimmunoglobulin molecules, but also anti-idiotypic antibodies, mutants,fragments, fusion proteins, humanized proteins and modifications of theimmunoglobulin molecule that comprise an antigen recognition site of therequired specificity.

An “antibody complex” is the combination of antibody and its bindingpartner or ligand.

A “native antigen” is a polypeptide, protein or a fragment containing anepitope, which induces an immune response in the subject.

The term “isolated” means separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.As is apparent to those of skill in the art, a non-naturally occurringpolynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, does not require “isolation” to distinguish it from itsnaturally occurring counterpart. In addition, a “concentrated”,“separated” or “diluted” polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, is distinguishable from its naturallyoccurring counterpart in that the concentration or number of moleculesper volume is greater than “concentrated” or less than “separated” thanthat of its naturally occurring counterpart. A polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, which differs fromthe naturally occurring counterpart in its primary sequence or forexample, by its glycosylation pattern, need not be present in itsisolated form since it is distinguishable from its naturally occurringcounterpart by its primary sequence, or alternatively, by anothercharacteristic such as glycosylation pattern. Although not explicitlystated for each of the inventions disclosed herein, it is to beunderstood that all of the above embodiments for each of thecompositions disclosed below and under the appropriate conditions, areprovided by this invention. Thus, a non-naturally occurringpolynucleotide is provided as a separate embodiment from the isolatednaturally occurring polynucleotide. A protein produced in a bacterialcell is provided as a separate embodiment from the naturally occurringprotein isolated from a eukaryotic cell in which it is produced innature.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agent,carrier, solid support or label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'SPHARM. SCI, 15th Ed. (Mack Publ. Co., Easton (1975)).

As used herein, the term “inducing an immune response in a subject” is aterm well understood in the art and intends that an increase of at leastabout 2-fold, more preferably at least about 5-fold, more preferably atleast about 10-fold, more preferably at least about 100-fold, even morepreferably at least about 500-fold, even more preferably at least about1000-fold or more in an immune response to an antigen (or epitope) canbe detected (measured), after introducing the antigen (or epitope) intothe subject, relative to the immune response (if any) beforeintroduction of the antigen (or epitope) into the subject. An immuneresponse to an antigen (or epitope), includes, but is not limited to,production of an antigen-specific (or epitope-specific) antibody, andproduction of an immune cell expressing on its surface a molecule whichspecifically binds to an antigen (or epitope). Methods of determiningwhether an immune response to a given antigen (or epitope) has beeninduced are well known in the art. For example, antigen specificantibody can be detected using any of a variety of immunoassays known inthe art, including, but not limited to, ELISA, wherein, for example,binding of an antibody in a sample to an immobilized antigen (orepitope) is detected with a detectably-labeled second antibody (e.g.,enzyme-labeled mouse anti-human Ig antibody). Immune effector cellsspecific for the antigen can be detected any of a variety of assaysknown to those skilled in the art, including, but not limited to, FACS,or, in the case of CTLs, ⁵¹CR-release assays, or ³H-thymidine uptakeassays.

By substantially free of endotoxin is meant that there is less endotoxinper dose of cell fusions than is allowed by the FDA for a biologic,which is a total endotoxin of 5 EU/kg body weight per day.

By substantially free for mycoplasma and microbial contamination ismeant as negative readings for the generally accepted tests know tothose skilled in the art. For example, mycoplasm contamination isdetermined by subculturing a cell sample in broth medium and distributedover agar plates on day 1, 3, 7, and 14 at 37° C. with appropriatepositive and negative controls. The product sample appearance iscompared microscopically, at 100×, to that of the positive and negativecontrol. Additionally, inoculation of an indicator cell culture isincubated for 3 and 5 days and examined at 600× for the presence ofmycoplasmas by epifluorescence microscopy using a DNA-bindingfluorochrome. The product is considered satisfactory if the agar and/orthe broth media procedure and the indicator cell culture procedure showno evidence of mycoplasma contamination.

The sterility test to establish that the product is free of microbialcontamination is based on the U.S. Pharmacopedia Direct Transfer Method.This procedure requires that a pre-harvest medium effluent and apre-concentrated sample be inoculated into a tube containing tryptic soybroth media and fluid thioglycollate media. These tubes are observedperiodically for a cloudy appearance (turpidity) for a 14 dayincubation. A cloudy appearance on any day in either medium indicatecontamination, with a clear appearance (no growth) testing substantiallyfree of contamination.

Examples Example 1. Development of Ghost Cells for Numerical andTargeted Expansion of T Cells, NK Cells, B Cells, and/or Other ImmuneCells or for Infusion to Treat Cancers and Autoimmune Diseases

“Ghost cells” are immortalized cell lines genetically modified toexpress immune co-stimulatory molecules anchored to lipid rafts via aglycosylphosphatidylinositol anchor (GPI) that is added to the proteinduring ER post-translational modification. The term “ghost cell” resultsfrom the preparation method to render the cell empty and non-viable.Ghost cell lines will undergo a specific hypotonic lytic procedure thatwill extract all intracellular components, but allows plasma membrane,GPI anchored proteins, and lipid rafts to remain intact. Hence,rendering the components “ghosts”. Ghost cells are used for thenumerical and targeted expansion of canine, human, feline, and equinelymphocytes through the placement of specific immunoediting geneticpressures on lymphocytes. The use of GPI anchors, use of lipid rafts,use of various species (human, canine, feline, and equine), applicationof immunoediting pressures for expansion, and use of a specifichypotonic lytic procedure constitute new art to which the inventionpretains. Ghost cells are a human malignant cell line that have beengenetically modified to express T-cell human molecules including, butnot limited to, CD137L, CD86, CD64, HLA-Cw 3, and HLA-Cw 5. Theco-stimulatory molecules will be attached to the ghost cell membraneusing glycosylphosphatidylinositol anchors (GPI). The GPI anchoring willensure that the transfected genes are expressed on the cell surface inclose proximation inside lipid rafts, rather than expressed randomlyacross the cell plasma membrane. A self-inactivating transposase andtransposon system or a drug selection equipped DNA printed plasmid willbe used to introduce the genes of interest via lipofectaminetransfection. Human cell lines, such as K562, 722.21, Jurkat or otheradherent/suspended cell line will serve as the basis for the ghostcells. The term “ghost cell” results from the method of preparing thecells to stimulate NK and T cell expansion such that they are empty andnon-viable. Ghost cell lines developed and tested will undergo aspecific hypotonic lytic procedure that will extract the intracellularcomponents, but will leave the cell membrane and lipid rafts intact,therefore, rendering them ghosts.

(I) Development of Plasmids that Contain T-Cell Co-Stimulatory Moleculesand HLA-Cw*3 and 5 Molecules

DNA plasmids and vectors will be created either using DNA printing, 3Dprinting, bacterial transformation, and/or other methods. Plasmids andvectors will be codon optimized for the mammalian cell species utilizedand will incorporate species specific promoters such as, but not limitedto CMV, EF1-alpha, CD3, and KIR. Genes of interest will be printed orcloned either into (i) a transposon vector with an associatedtransposase to cleave the gene of interest from the transposon andinsert it at specific repetitive sites into the cell's or (ii) a type ofviral vector that can be used to transduce a cell randomly with the geneof interest to cause protein expression. DNA or FIG. 2B shows possibleplasmid maps.

(II) Isolation of Canine Peripheral Blood Mononuclear Cells (PBMC)

Canines diagnosed with an applicable malignancy will have peripheralblood collected before SOC treatment or an apheresis collected postNeupogen dosing and prior to HSCT. The apheresis volume range for HSCTis 120 to 350 ml. The volume collected depends on size (weight) of dogand is collected on a TerumoBCT Spectra or Optia machine. If apheresisis not possible, 50 to 150 mls of peripheral blood will be drawn viaintravenous catheter in an extremity chosen by the attendingveterinarian into a blood bag designated for clinical use. For smallervolumes, EDTA vacutainers may be used. Some veterinarians may elect topre-treat the patient with Neupogen prior to peripheral blood collectionto increase PBMC numbers. The sample will be overnighted to a certainfacility at room temperature, unless the daytime temperatures are above85 F. Then, the sample should be sent with an ice pack to maintain cellviability and status. Upon arrival at the facility, the patient samplewill receive a unique number identifier that will be linked to thepatient's attending veterinarian's office client number. At this time,data from the patient information sheet will be entered into thedatabase. The sample will be decanted and diluted with Hank's BalancedSalt Solution (HBSS) at a ratio of 1:5 (sample:HBSS). Samples will beoverlayed onto Ficoll at a ratio or 1:2 (Ficoll:diluted sample) in 50 mlcentrifuge tubes and centrifuged for 40 minutes at 400×g with no brake.The PBMC layer will be removed using sterile, disposable transferpipettes and diluted 1:1 with HBSS before centrifugation at 450×g for 15minutes with medium brake. Supernatant will be discarded and the cellswill be diluted again with HBSS at a 1:1 ratio before centrifugation at400×g for 10 minutes. Fifty percent of the PBMCs will be cryopreservedas back up, 45% will be used for expansion on ghost cells, and 5% willbe phenotyped by flow cytometry to provide a patient baseline and placedin their chart.

(III) Canine, Feline, Human, or Equine PMBC, T-Cell, and Infusion SampleCryopreservation

Canine, feline, human, or equine PBMCs after isolation will becryopreserved in in-house freeze media (FM) or commercially derived FM.This media will be made in batches with accompanying recorded batchnumbers, aliquoted and frozen until use, and undergo sterility testingfor bacteria, fungus, mycoplasma, and endotoxin for quality control. FMmay contain the following solutions: 40-70% Saline (PBS, HBSS, etc),20-40% canine, feline, human, or equine serum, 1-10% Dimethyl sulfoxide(DMSO) and may be filtered sterilized one to three times. The serum willbe heat inactivated at 65 C for 30-60 minutes to inactivate thecomplement proteins before aliquoting and freezing until use. The serumwill be purchased from regulatory approved sources. DMSO and HBSS willbe purchased commercially. After centrifugation and removal ofsupernatant, T cells will be resuspended in thawed (4 C temp) FM at aconcentration range between 5×10⁶ to 1×10⁸ cells/ml. In the instances ofback-up cryopreservation at isolation and after stimulations, cells willbe cryopreserved in 2-10 ml cryovials using isopropanol containingcanisters in −80 to −65 C for 24-72 hrs before transfer to liquidnitrogen storage for permanent storage. Samples for patient's infusionswill be cryopreserved in bags with ports suitable for cryopreservationand infusion. Samples earmarked for infusion may be cryopreserved usinga rate-controlled freezer for 45-52 minutes using a specific program andthen will be transferred to liquid nitrogen for storage until infusion.Twenty-four hours prior to infusion, the T cells will be sent to theveterinarian on dry ice in Styrofoam and cardboard container overnightwith a copy of the infusion protocol, follow-up instructions, andresults of the T-cell quality control and assurance. Samples will betested flow cytometrically for lack of B cells (less than 5%), majorityT cells (greater than 50%), CD4 and CD8 percentages, viability, lack ofghost cells (less than 5%), and presence of the TCR or CAR iftransfected (greater than 10% positive).

(IV) HLA-Cw*3 and/or 5 Positive Ghost Cell Transfection,Cryopreservation, and Formation

Human cells lines (either adherent or in suspension) expressing HLA-C*3and/or 5 will be transfected with genes of interest (human CD86, CD64,CD137L, and IL-15 (all extracellular domains will be expressed on GPIanchors)) embedded in a piggybac transposase/transposon system. Geneswill be transfected using the DMRIE-C reagent which is commercially soldby Invitrogen. This protocol will be optimized for each cell line andgene. If the human cell line does not endogenously express the proteinHLA-C*3/5, then it will be transfected into the cell line as well usingthe piggybac technology. Cell lines to be developed into ghost cells mayinclude, but will not be limited too, K562, 722.21, and Jurkat. Mastercell banks of each line prior and post stable transfection will becreated and stored in liquid nitrogen. Prior to transfection, cell linesmay undergo cell line fingerprinting to ensure the correct identity ofthe line. Cell lines will be expanded at 39° C. in a humidifiedincubator using complete media (CM: RPMI, 10% 100× glutamax, 10% fetalcalf serum (FBS)). Cell lines will be cryopreseved in FM (10% DMSO, 50%HBSS, 40% FBS) at a concentration of 2 to 4×10⁷ cells/ml in 2 mlcryovials. Transient transfection will be determine 24 hours posttransfection using flow cytometry and antibodies recognizing theinserted gene's protein expression. Single color and no stained controlswill be used to aid in color compensation and expression analysis. Ghostcell lines will receive 50% to 100% media changes 2 to 3 times a week tomaintain the cellular concentration at 2×10⁶ cells/ml of CM. Stableexpression will be measured seven days post transfection utilizing the24 hour measurement protocol and analysis. Positive expressing cellpopulations will be sorted using antibodies and magnetic columns orusing Influx cell sorter. The positive fraction will be expanded asoutlined previously and will be sorted weekly or bimonthly until all ofthe cells express all inserted genes. Quality control and assurance willbe completed on all master and working ghost cell banks (Sterility,Endotoxin, Mycoplasma, Viability, Gene Expression, Fingerprinting).

Ghost cells will be formed using hypotonic lysis prior to co-culturewith T-cells. The hypotonic lysis solution will contain cocktails toinhibit protein degradation (tyrosine, acid and alkaline phosphatases;serine proteases; trypsin, chymo-trypsin, plasmin, and amino-peptidases;cystein proteases; L-isozymes, and serine-threonine proteinphosphatases) diluted in distilled water with a pH of 7.0. Ghost celllines will be incubated for 5 to 14 minutes, depending on the ghost cellline.

More specifically, to create a ghost cell for immune cell activation or“ghostbusting”, a ghost cell trainer (e.g., transfected cell line) aretransferred to 10-250 ml centrifuge tube and centrifuged at 400G for 5to 12 minutes. Supernatant will be removed and the ghost cell trainercells resuspended in saline before quantification and viabilitymeasurement using AO/PI. Ghost cell trainer cells will be washed,centrifuged using the same technique and supernatant removed. Ghost celltrainers will be resuspended in the above described hypotonic solutioncontaining a protease inhibitor for period of time (1 to 14 minutes)depending on the ghost trainer cell number (1e6 to 1e9 cells/mL ofhypotonic lysing solution). Ghost cells will be centrifuged at 400 to2500 g for 3 to 15 minutes. Supernatant will be removed and viabilityassessed using standard AO/PI cellometer imaging.

Viability must be less than 30% before co-culture with immune cells.Ghost cells will be resuspended in the appropriate immune cell completemedia in 5 mLs.

Viability and cell size will be assessed using trypan blue as measuredby a cellometer post incubation and washing with RPMI media containingprotease inhibitor cocktails at 200×g for 10 minutes. Solutions withcell pellets/lysates less than 20% will immediately prepared forco-culture. Membrane integrity, protein expression and lipid raftintegrity endurance will be assessed using antibodies and single quantumdots connected to a cholera toxin B subunit for microscopicvisualization and quantification. Cholera toxin B subunit it a moleculethat binds to ganglioside, GM1, which is a major mammalian cell lipidraft constituent. This visualization will allow optimization of theincubation period, as well as, molarity of the solution by determiningthe optimal number of lipid rafts necessary for canine T-cellactivation. We will correlate this data with viability and include itour quality control ghost cell panel.

(V) Canine T-Cell Expansion on Ghost Cells

Canine T cells will be co-cultured with HLA-Cw*3 and 5 positive ghostcells to expand a TCR-specific oligoclonal T-cell population thatrecognizes HLA-Cw*3 and 5 presented antigens, such as Importin subunitalpha. Canine T cells will be expanded in a 39 C humidified incubator inCM (RPMI, 1-100% 100× glutamax, 1-100% FBS) at a concentration of 1.5 to2×10⁶ T cells/ml of CM. Exogenous human cytokines (IL-21 and IL-2) willbe added to the cultures 2 to 3 times per week at a concentration ofand, respectively. IL-2 will not be added for the first 7 to 10 days. Tcells will be re-stimulated with ghost cells every 4 to 8 days until aclinically sufficient number of T cells has been reached for eachpatient. This number is based on body surface area (BSA) calculated fromthe weight of the dog. Ten percent of every other stimulation will becryopreserved as back-up. Ghost cells will be added to the T-cellculture at a ratio ranging from (Ghost Cell: T-cell) 1:1 to 1:3. Theexact ratio will be determine through optimization experiments as thismethod of stimulation vastly differs from the addition of viable, butgamma-irradiation apoptotically-induced artificial antigen presentingcells. T cells may be phenotyped flow cytometrically for CD3, CD4, CD8,CD21, CCR7, Granzyme B, Perforin, IFN-g, and CD32, among others at days7-47 and prior to cryopreservation for infusion. Cells may be expanded 7days or greater and we expect that not all patient samples will expandsimilarly or at all. All cultures may be checked weekly for mycoplamsaand endotoxin.

(VI) Infusion Protocol for Veterinarians

1.) Sterilely place intravenous catheter and T-port in patient. Have allsupplies ready; 2.) Remove frozen product/unit. Seal in clean waterimpermeable plastic bag; 3.) Place unit/sealed bag into warm water bathor bowl of water (37° C.); 4.) Gently rock bag in warm water until fullythawed; 5.) Remove product/unit from sealable bag; 6.) With clean/drygloves sterilely place filtered bag access spike; 7.) Gently withdrawentire contents of bag into 60 ml syringe with 18 g needle; 8.) Instill10-20 ml sterile 0.9% NaCl+10 ml air into bag/unit from pre-drawnsyringe; 9.) Gently mix bag and withdraw contents into 60 ml syringefrom above; 10.) Slowly administer product over 10 minutes (5 ml/minutemaximum rate); and 11.) Flush IV catheter with pre-drawn 5-10 ml ofsterile 0.9% NaCl.

Check and record the following: temperature, heart rate, respiratoryrate, and reactions at 5, 10, and 30 minutes post T-cell infusion, aswell as, pre-infusion. This is to monitor for any immune reactions thatmay occur. If an adverse reaction is recorded, CAVU Biotherapies must benotified in writing and a 10 ml peripheral blood and serum sample at thetime of the reaction must be collected and sent to CAVU Biotherapies forimmune analysis. The following supplies are necessary for each infusion:standard intravenous catheter 20-22G, T-port, #1 60 ml leur-lok syringe,#1 30 ml leur-lok syringe, #1 10 ml leur-lok syringe, #3 18G needles,new sterile 0.9% NaCl bag, Hemo Tap Filter (Bag Access) Spike, warmclean water bath or preheated warm water in clean bowl (370 C), sealableplastic bag, Nitrile gloves, and a dedicated thermometer.

The time from start of thaw to completion of the T-cell infusion is 15minutes (max 30 minutes). After 15 minutes, the viability of theinfusion begins to diminish. Other information necessary for clinicians:pre-place needles onto syringes; pre-draw 10-20 ml 0.9% NaCl+10 ml air;volume of flush at doctor preference; pre-draw 5-10 ml sterile 0.9% NaClfor post infusion IV flush; insure bag/unit and access ports remainclean and dry at all times; and wear gloves at all times.

(VII) Osmolytic Creation of Ghost Cells

To create a ghost cell for lymphocyte expansion or “ghostbusting”, atransfected cell line will be subjected to a hypotonic solutioncontaining a protease inhibitor for an optimized period of time (1 to 5minutes). This pore forming solution will cause the initiation ofcaspase 3, 8 and mitochondrial apoptotic cascade, as well as, create alarge hole in the weaker side of the cell membrane to allow the entireintracellular contents to leak out. Trypan blue and the cellometer willbe utilized to gauge viability every 12 to 24 hours for a period of 72hours. During the first 6 hours, ghost cells will be monitored forlysis, membrane integrity, and apoptotic cascade initiation.Optimization of hypotonic solution recipes and incubation periods willbe completed at this time. While the cell will be dead, the cellmembrane with the lipid rafts will remain relatively intact to allowT-cell binding. Hence, the origination of the term “ghost cell”.Hypotonic stress initiates apoptotic pathways rather than necroticpathways, which are more advantageous for T-cell activation (Selzner2004). Hypotonic stress causes cell death through a caspase andmitochondrial-dependent mechanism, as well as, up-regulates GPI proteinand MHC expression. Volume sensitive ATP release plays a key role in theinduction of apoptosis following hypotonic exposure. This is a novel wayof inducing T-cell activation as current methods use more expensive andregulatory complex cesium irradiation to kill the tumor cell lines. Thisirradiation method allows the tumor cell lines continue to function ashighly viable artificial antigen presenting cells in culture for daysafter irradiation. These artificial antigen presenting cells remainintact and have the capacity to proliferate, move, and secreteimmunosuppressive cytokines. Ghost cells remove the immunosuppressivecytokine secretion (IL-10 and TGF-B), prohibit tumor cell lineclustering to inhibit T-cell mediated lysis, prohibit T-cell toleranceto tumor antigens, and prohibit possible tumor proliferation andviability, while improving synaptic formation and strength to increaseT-cell expansion.

Example 2. Antigen-Specific TCR HLA-Cw*3 and 5 Driven Autologous andAllogeneic T-Cell Expansion on Ghost Cells

T cells will be co-cultured with these ghosts 1 to 3 times per week withthe addition of exogenous human IL-2 and IL-21 cytokines, 2 to 4 timesper week. Once clinically sufficient numbers of HLA-Cw*3 and 5 tumortargeted T cells are expanded, cells will undergo quality control andassurance after cryopreservation for infusion. TCR sequencing will becompleted on optimized cultures to support oligclonal and TCR-specificT-cell growth.

By targeting HLA-Cw*3 and 5 TCR recognizing T cells, we will expand anoligoclonal TCR population recognizing TAAs, like Importin, which arepresented on this specific molecule. This will differ from past methodswhich relied on OKT3-driven canine T-cell expansion, which drivespolyclonal non-specific expansion. By targeting TAAs expressed throughHLA-Cw*3 and 5, we will be expanding a TCR-specific T cells. HumanHLA-Cw*3 and 5 cross reacts with several animal species, specificallyporcine (cited), and there is a 91% homology between the human andcanine when the sequences are BLASTed against each other.

To drive TCR-specific expansion, ghost cells will be hypotonically lysedas described above and centrifuged immediately before addition thePBMC/T cells. Ghost cells will be added at a ratio (ghost cell to Tcell) from 1:2 to 1:4, once to three times per week. Exogenous humancytokines (IL-2 and IL-21) will be used to accelerate the desired CD8 orCTL T-cell expansion. Concentrations will be optimized to create optimalexpansion and activation with ghost cells and lipid rafts, but willstart at 2 to 3 times per week dosing. Canine T cells and Ghost cellswill be grown in a humidified incubator at 39 C, which is approximatelythe ambient temperature of a normal dog. T cells will be weeklyenumerated using trypan blue and cellometers, phenotyped (CD3, CD4, CD8,CD25, CD21, CCR7) using flow cytometery and viability analyzed to ensureclinically sufficient numbers of CTLs can be produced. Prior tocryopreservation, T cells will be tested for granzyme B and IFN-γproduction.

TCR will be sequenced on 6 different expanded patient samples to showTCR oligoclonal population distribution. Canine T-cell gene specificNanostring panels may be employed in 12 patient samples (pre and postexpansion) to show that T-cell dysfunction has been overcome using ghostcell manipulation. For cryopreservation purposes, a quality control andassurance protocol will be developed that measures viability by trypanblue, phenotype (CD3, lack of ghost cells, lack of B cells), sterility(bacteria, fungal), mycoplasma, and endotoxin levels. This will becompleted on all infusion samples. Infusion samples will becryopreserved in cryopreservation bags using an in-house produced andquality tested freezing media (10% DMSO, 50% HBSS, 40% Canine Serum fromNorthwest Blood Bank) and frozen to −100° C. within 3 hours of finalharvest. Freezing will take place using a Rate Controlled Freezer andthe bags will be immediately transferred to liquid nitrogen for storage.

Example 3. TCR Sequencing on T Cells Expanded on Other Ghost Cell Linesto Identify New TCR Sequences and Targets

Participating veterinarian oncologists and veterinarians will routinelytest for relapse using clonality testing, pathology, andimmunophenotyping of the peripheral blood and suspicious growths.Peripheral blood and fine needle aspirates of the complying caninepatients will be submitted to O'Connor Immunogenetics to determinedominant TCR clone analysis using next generation sequencing.

Companion canines will be clinically for relapse by participatingveterinarians and veterinarian oncologists. PCR or clonal testing onperipheral blood samples will determine B-cell lymphoma relapse. Tumorpathology or imaging may be completed to determine reoccurrence in othermalignancies. Along with clonal testing and pathology, peripheral bloodT-cell phenotypes (CD3 numbers, CD3CD4:CD3CD8 ratio numbers) will beroutinely (monthly or every other month) monitored to determine if thereare changes (decreased CD3 numbers, increased CD3CD4:CD3CD8 rationumbers) that suggest relapse.

In those companion canines that respond spontaneously, peripheral bloodor tumor infiltrating T cells at these time points will be collectedthrough 10 ml blood draws and/or fine needle aspirates (FNA). T cellswill be separated using a ficoll density centrifugation protocolestablished in Specific Aim #1. The PBMC will be submitted for TCRsequencing using 25-30 nucleotide primers for use on MiSeq technology.

The dominant TCR sequences observed from in-house bioinformatic analysiswill determine if the infused T cells have persisted and developed intoa memory state or if the tumor has antigenically mutated due toimmunoediting and another tumor-targeting TCR has developed. Massspectrometry and protein isolation techniques will be employed todiscover the new TAA. Resulting data will be used for the potentialdevelopment and generation of alternative TAA TCR-specific T cells thatmay or may not be genetically modified for relapsed canines and thesedata/sequences may be licensed for human use.

Example 4. HLA-CW*3/5 Positive T Cell and NK Cell Infusion SpecificDosing Regimens

Standard of care therapies may include CHOP, abbreviated CHOP, tumortargeted gamma-irradiation, surgery, and autologous or allogeneichematopoietic stem cell transplantation (HSCT). Pre-infusionconditioning treatments will include high dose cytoxan and possibletotal body irradiation (TBI) at a dose of less than 2 Gy.Immunomodulating treatments not only provide efficacious treatment whilethe cellular therapy is being prepared, but can provide a suitableenvironment for the infusion to expand, lyse targets, and engraft.

The following treatments may be used as disease-stabilizing orremission-inducing treatments while the T-cell are being manufactured:CHOP, abbreviated CHOP, MOPP, doxil, doxorubicin, tumor-targeted gammairradiation, surgery, prednisone and autologous or allogeneichematopoietic stem cell transplantation (HSCT) or donor lymphocyteinfusions (DLI).

Pre-infusion conditioning treatments will include high dose cytoxan andpossible total body irradiation at a dose of less than 2 Gy.Immunomodulating treatments not only provide efficacious treatment whilethe cellular therapy is being prepared, but can provide a suitableenvironment for the infusion product to expand, lysed targets, andengraft. Pre-conditioning treatments remove immunosuppressive cells andcytokines, modulate the tumor and its microenvironment, removes singlecell metastases, and creates space in the lymphoid compartment for theex vivo expanded HLA-Cw*3 and 5 TAA recognizing T cells.

In companion canines diagnosed with OS, tumors will be treated with 2fractionated doses of 8 Gy to equal a total dose of 16 Gy over 48 to 72hours. Forty-eight hours post the final dose of radiation, the firstT-cell dose of 5×10⁸ cells/m² will infused intravenously. The patientwill be monitored for 1.5 to 3 hours post infusion for any adversereactions. Seven days post the first infusion another T-cell sample of5×10⁸ cells/m² will be administered intravenously. A total of two tothree T-cell infusions may be given. Swelling at the primary andmetastatic sites may occur, as well as, in the draining lymph nodes.Imaging (may include X-ray, Ultrasound, CT scan, MRI scan) may becompleted prior to therapy and at all follow-up appointments as deemednecessary by the veterinarian. This data will be collected.

In companion canines diagnosed with B-cell LSA, the disease may betreated with SOC therapy. A minimum of 10 to 14 days post SOC must passbefore pre-conditioning with high dose cytoxan occurs. Seven to ten dayspost pre-conditioning treatments, B-cell LSA diagnosed canines will beinfused intravenously with one dose of 5×10⁸ cells/m². Fourteen dayslater the patient will be infused intravenously with a second dose of5×10⁸ cells/m². B-cell LSA patients will receive 2 T-cell doses afterthe initial SOC treatment. The patient will be monitored for 1.5 to 3hours post each infusion for any adverse reactions.

Companion canines diagnosed with AML have an extremely short time beforeblast crises develops. Current SOC is autologous or allogeneic HSCT withhigh dose cytoxan treatment at relapse. Fourteen days post the last doseof cyclosporine after the HSCT or 7 to 10 days post high dose cytoxantreatment (if relapse occurs), the first T-cell infusion will occurintravenously at a dose of 2×10⁸ cells/m². Dogs with AML will receive aT-cell dose of 3×10⁸ cells/m², then 4×10⁸ cells/m², and then at amaximum dose of 5×10⁸ cells/m² every 7 to 10 days post the priorinfusion for a total number of infusions of up to 8 infusions dependingon the patient's response and toleration of side effects. Blasts will bemonitored 1 to 2 times per week to monitor efficacy and immuneactivation against the tumors.

In companion canines diagnosed with HS, SOC therapy will be initiatedwhile T-cells are being manufactured. Seven to ten days postpre-conditioning high dose cytoxan treatment, the first T-cell infusionwill occur intravenously at a dose of 2×10⁸ cells/m². Dogs with HS willreceive a T-cell dose of 3×10⁸ cells/m², then 4×10⁸ cells/m², and thenat a maximum dose of 5×10⁸ cells/m² every 7 to 10 days post the priorinfusion for a total number of infusions of up to 4 infusions dependingon the patient's response and toleration of side effects.

Allogeneic T-cell infusions will only in occur in a setting postallogeneic HSCT or DLI. Allogeneic T-cell infusions will not occur alonewith any other type of SOC treatments or treatments of experimentalnature for safety reasons to prevent lethal graft versus host disease(GVHD).

Example 5. Biomarkers Panels and Immunoscores to Identify Relapses,Anti-Tumor Immune Response, and Immune Status

These panels may include sequencing analysis, cytokine secretion, PCR,and flow cytometric analysis, which can be combined to predict futureresponses using machine learning models and subsequent, algorithms,bioinformatics, and scripts. Machine learning based predictive computermodeling scripts will be generated and optimized on free statisticalprograms like “R”, or through python or artificial intelligence-basedsystem. Patient gene and protein data will be parsed into a computermodel training set that will be used to train the computer to predictimmune responses, potential efficacy, and adverse events. These datawill be transferred by a bioinformatic specialist into an accessibleformat that can be used by clinicians to improve patient quality of lifeand outcomes, while personalizing the patient's treatment protocol.

These panels may include sequencing analysis, cytokine secretion, PCR,and flow cytometric analysis, which can be combined to predict futureresponses using machine learning models and subsequent, algorithms,bioinformatics, and scripts.

(I) Healthy Baseline Determination and Development of Immune SystemPanel

Serum and peripheral blood (5 ml and 10 ml, respectively) will becollected from 100 normal/healthy companion canines (all breeds, ages 2to 5 years) at their yearly wellness exams across the United States toprovide a necessary cross-section and baseline of canine immune status.Cytokine serum levels (IL-6, TGF-B, IL-10, IL-2, IFN-γ, etc) will betested via ELISA or Luminex technologies. Peripheral blood will beanalyzed using flow cytometry to determine CD3 T-cell numbers, CD3CD4T-cell numbers, CD3CD8 T-cell numbers, CD4CD25 T-cell numbers,CD20/21/19 B-cell numbers, Granzyme B, perforin, and ratios. Copies ofthe CBCs from the primary veterinarian will also be collected todetermine the absolute T cell counts and neutrophil to lymphocyteratios, as well as, other data necessary to derive a complete picture ofthe canine's normal immune system. Canine-specific Nanostring panels orPCRs may be employed to determine intracellular protein expressions andsignaling pathway activations. Testing will include ZAP70, CD3-zeta, andexpression. TCR sequencing may be completed on 5 to 10 healthy caninesto determine TCR repertoire changes and how these relate to the immunehealth of the canine before diagnosis of cancer. These protocols will bedeveloped to include quality control and assurance necessary forclinical use. Data will be analyzed using the R statistical program witha script that will outline ranges, medians, means, and standarddeviations. By combining all facets of this data using modeling (SectionB.5.4.), an “Immunoscore” will be created. This number will giveclinicians a numerical value that is representative of overall immunehealth.

(II) HSCT Transplant Immune Reconstitution

Serum and peripheral blood (5 ml and 10 ml, respectively) will becollected from 10 companion canines who have underwent a HSCT(autologous or allogeneic) across the United States to provide anecessary cross-section and baseline of canine immune reconstitution preand post transplant. This will allow one to fine tune adoptive cellularstrategies in these cases by understanding how the canine immune systemreacts to transplantation procedures in the private practice settings.Canine must have a diagnosis of B-cell LSA or AML and must be treated bylicensed veterinarians. Serum, CBCs and peripheral blood samples will becollected pre-transplant, 14 days post transplant. 28 days posttransplant, 42 days post transplant, 70 days post transplant and 108days post transplant. Data will be collected and analyzed as outlinedabove.

(III) Adoptive T-cell Transfer Reconstitution

Serum and peripheral blood (5 ml and 10 ml, respectively) will becollected from companion canines which have had a diagnosis of B-cellLSA, AML, OS, or HS across the United States to provide a necessarycross-section and baseline of canine immune reconstitution pre and postadoptive therapy. This will allow one to fine tune adoptive cellulartreatment strategies in these cases by understanding how the canineimmune system reacts to cellular infusions procedures in the privatepractice settings. Serum, CBCs, PCR and peripheral blood samples will becollected at diagnosis, 7 days post SOC therapy, 7 days postconditioning treatment (pre-T cell infusion), 7 days post each adoptiveT-cell infusion, and then at monthly rechecks as recommended by theoncologist. Data will be collected and analyzed as outlined above.

(IV) Machine Learning to Predict Immune System Behavior in Canines (andHumans) and Immunoscore Development

The increase of computer integration into patient's health has allowedthe collection of data which assists in patient treatment plans.However, medicine is still, in many ways, practicing reactive medicinerather than proactive medicine, i.e. anticipating the patient's medicalneeds. Evolutionary learning combines aspects of machine learning withbiological processes or natural evolution that are in constant flux.With dramatic interest in oncological immunotherapies, a looming,ever-present need is to predict how a vaccine or adoptive cellulartherapy using TCRs will influence the patient's systemic immunity andreactions. The immune system and its landscape evolves continuously andlearns based on endogenous and exogenous pressures similar to naturalselection. Here we show that a combination of statistical analysis andmachine learning of T cell gene signatures in companion caninesdiagnosed with B-cell lymphoma can predict with accuracy immune statesrelated to remission, relapse, and homeostasis. These data will allowscientists and clinicians a like to determine which patients willbenefit most from adoptive cellular therapy, when to infuse, dosingschemes, adverse events, predictions, and personalized treatments tobolster or dampened the resulting immune response. Metazoans developfunctionally distinct cell types using essentially the same genomicblueprint. Over its life cycle, each cell transitions through distinctstates (e.g. quiescence, differentiation, proliferation, senescence,etc) which are associated with manifestly distinct gene expressionprofiles (again, importantly transcribed from essentially the samegenomic content). Mammalian genomes, carrying approximately 25,000genes, impart an astronomical total possible combination of expressionpatterns and cell states. While molecular pathways, typicallyschematized as arrow-to-arrow diagrams (X->Y->Z), are usefulrepresentations of simple biochemical cascades, they lose clarity andsimplicity to embody the high complexity and density of genome-wideexpression data (“omics” data). Even by linking multiple pathways intocircuit architectures of gene regulatory networks (GRNs), the need forintegrative approaches stands. Not with-standing the complexity ofunderlying molecular interactions, integrative systems biology arerealized by applying predictive models (Huang 2012). The scriptsdeveloped for the Immunoscore gene expression prediction profiles arebased.

A website would be created so that clinicians and scientists couldupload their data on the cloud and patient information. Alternatively,patient samples would be sent to a validated wet lab for processing. TheCompany would then be able to directly access the data once the patientand clinician signed waivers to release the information. Clinicianscould choose the type of analysis needed. For a fee, we would analyzethe data using a cloud and specific programming scripts. The scriptswould encode analysis for Z scores, ANOVA, Pearson Correlation, PCAeigenvectors, and HMM or GLM analysis. This could be done HIPPAcompliant and would set the standard for analysis. The analysis would beprovided in an email or they could log on to the website. TheImmunoscore could predict prognosis and diagnosis using gene expressiondata, phenotype data, blood and serum protein levels, and imagingresults. The Immunoscore would be further broken down to includespecific genes or factors using PCA that contributed the greatestinfluence on the score. This information would supply clinical decisionsupport. Validation and calibration of the scripts and computer will beroutinely completed as a part of quality control.

Example 6. Tumor Antigen-Specific TCR and CAR Portfolios

New scFv sequences will be generated against canine tumor targets. Thestalk, hinge, transmembrane domain, and endodomains will be optimizedfor canine use. T cells will be transfected using a self-inactivatingtransposon and Transposase system.

The premise for this protocol is to exploit the SHM and AID in acontrived setting that would mutate the TCR within 24 hours andcorrectly identify tumor antigen and mutated TCR pairings. The TCR orscFv CAR would be sequenced and transfected into patient's T cells forexpansion and subsequent more efficacious infusions.

(I) Mechanisms of SHM

The dog and human Activation-Induced Deaminase (AID) nucleotide sequencehomology is 95% after BLAST-comparing. This supports the translationalcapabilities of the SHM method to develop new TCR and CAR tumor-antigenepitope recognizing sequences. Somatic hypermutation (SHM) is a cellularmechanism by which the immune system adapts to new foreign elements thatconfront it, as observed during class switching. SHM specificallydiversifies B-cell receptor (BCR) but, we believe can be harnessed inmanipulated setting to diversify the TCR as well. SHM involves aprogrammed process of mutations affecting the variable regions ofimmunoglobulin genes. Unlike germ line mutations, SHM affects only anorganism's immune cells and these mutations are not passed on to theoffspring. The BCR locus undergoes an extremely high rate of somaticmutations that is at least 10⁵ to 10⁶ fold greater than normal mutationrates that occur in the genome. Variations are mainly in the form ofsingle base substitutions in “hotspots” or hypervariable regions. Thesesites involved in antigen recognition on the immunoglobulin.

(II) Mechanisms of AID

Experimental evidence supports the view that the mechanism of SHMinvolves deamination of cytosine to uracil in DNA by AID. Acytosine:guanine pair is thus directly mutated to a uracil:guaninemismatch. Uracil residues are not normally found in DNA, therefore, tomaintain the integrity of the genome, most of these mutations must berepaired by high-fidelity DNA mismatch repair enzymes. The uracil basesare removed by the repair enzyme uracil-DNA glycosylase. Error-prone DNApolymerase are then recruited to fill in the gap and create mutations.The synthesis of this new DNA involves error-prone DNA polymerase, whichafter introducing mutations either at the positions of deaminatedcytosine itself or neighboring base pairs. During B-cell division andactivation, the immunoglobulin variable regions is transcribed andtranslated. The introduction of mutations in the rapid proliferatingpopulation of B cells ultimately culminates in the production ofthousands of B cells, possessing slightly different receptors or varyingspecificities for the antigen, from which the BCR with the highestaffinities for the antigen can be selected (Li, Wool et al. 2004;Janeway, Travers et al. 2005; Odegard and Schatz 2006; Teng andPapavasiliou 2007; Liu and Schatz 2009).

(III) Fast-Track TCR/CAR Portfolio Development

To build the TCR and CAR portfolios in a fast-tracked manner, autologousor allogeneic activated T fcells will be transfected with a 3D-printedlinearized DNA plasmid of AID using lipofectamine. These cells will beincubated briefly in a fluorescent dye that will emit light when themutated TCR/CAR binds to a tumor antigen. The AID+ve T cells will beco-cultured with ghost cell lines, autologous tumor or other cell lines.Three to 24 hours post co-culture at a 1:1 to a 1:4 ratio, fluorescent Tcells will be harvested, sorted by fluorescence, and the positiveTCRs/CARs will be sequenced using a commercially available MiSeqtechnology. Due to the large number of data collected per sample,sequence analysis will be carried out through rental of cloud space andtime on the AWS server. The analyzed sequences will be then added tointo a DNA plasmid that can be readily transfected into human and canineT cells. The mutated TCRs/CARs will be tested to ensure target-specifickilling by granzyme B, perforin, CD107a and IFN-γ production; andfluorescent killing assays with cell lines that are target antigennegative and positive. Data will also be collected on expansion ratesand weekly phenotype expressions. Genotype data will also be collectedfor mRNA expression analysis to determine correct T-cell function.

(IV) Patient Treatment with Mutated TCR/CAR

T cells from an oncology patient will be collected from aphaeresis orperipheral blood as described previously. The T cells will be activatedon ghost cells with IL-2 or IL-21 that have been transfected with themutated TCR or mutated CAR. Once the number of T cells reaches aclinical number, the T cells will be harvested and cryopreserved forin-process testing and infusion as previously described.

Example 7. Immunostimulation or Depression Through the Infusion of GhostCells

In both cancer and autoimmune diseases, a systemic immunologicaldysfunction has occurred. Previous T-cell therapies for lymphoma andleukemia in humans have shown the varying degrees of infused CAR/TCRpositive T-cell persistence which correlate with overall survival. Also,the infusion of T cells with persistence can produce long-lastingeffects and correct the immunodysfunction in dogs with cancer.

Using the immunoscore (a combination of ANOVA, Pearcon Correlations,Z-scores, 3D PCA, and Hidden Markov Model), it is evident thatimmumodulation occurs. Therefore, I propose creating ghost trainer cellsthat only express on the cell surface a combination of GPI-linkedcytokines and 1 type of MHC molecule. The trainers may express acombination of IL-10, TGFbeta, TNF-alpha, IFN-gamma, IL-21, IL-15, IL-2,IL-4, IL-12, IL-18, IL-6. The MHC will present a known auto-antigen inthe case of the autoimmune diseases. Pro-inflammatory “Uppers” andanti-inflammatory “Downers” cytokine ghost trainers would be created aspreviously described. The cytokines and MHC (for autoimmune only) wouldbe expressed in lipid rafts. Cells would be ghosted as earlier describedand infused intravenously into human, feline, canine, or equine patientssuffering from cancer, GVHD, or an autoimmune disease. The infused ghostcells would act as an adjuvant to suppress or activate systemic immunityvia T-cell, NK cells, and B-cells. For autoimmune diseases, the MHCwould express a known auto-antigen that would attract the dysfunctionalT cells to the raft where it would bind and undergo suppression orapoptosis from the cytokine signaling. For cancer, the pro-inflammatoryghost would create a systemic immune activation in response toneo-antigens expressed after chemo or radio therapy. Dosing would bebased on body surface area and weight. Infusions would take place duringan active cycle of the autoimmune disease or GVHD, while for cancer theghosts would be infused 24 hours to 7 days post chemo or radio therapyor at the first sign of immunosuppression. Dosing would start at 1e7cells/m² to 5e9 cells/m². Patients would receive unlimited doses,depending on tolerance, efficacy, and clinician's opinion.

Example 8. Exemplary Sequences Useful in the Production of Ghost CellTrainers or Car Cells

CMV promoter (www.algosome.com/resources/common-sequences.html#cmv) asshown below:

(SEQ ID NO: 1) TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAG. Kozack consensus sequence:(SEQ ID NO: 2) GCCGCCGCCATGG.

CD64 (NM_000566.3, P12314): signaling peptide: 1-15; extracellular:16-292 as shown below:

(SEQ ID NO: 3) QVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYIVIGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFH

CD86 (NM_175862.4 variant 1, isoform 1, P42081): signaling peptide:1-23; helical TM: 248-268; cytoplasmic: 269-329; extracellular peptides:24-247 as shown below:

(SEQ ID NO: 4) APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP

CD137L (NM_003811.3, P41273): cytoplasmic domain: 1-28; helical, signalanchor for type II membrane protein TM: 29-41; extracellular domain:50-254 as shown below:

(SEQ ID NO: 5) ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSE

HUMAN IL-15 (P40933) as shown below: signal peptide: 1-29; propeptide:30-48; chain: 49-162

(SEQ ID NO: 6) MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS

CD55 PROPEPTIDE AND LIPIDATION GPI SIGNALING ANCHOR, PROPEPTIDE: 353-381as shown below:

(SEQ ID NO: 7) TTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

NM_000574.4, Sig peptide: 295-396 as underlined; Mat peptide: 397-1353as italicized:

(SEQ ID NO: 8)    1agcgagctcc tcctccttcc cctccccact ctccccgagt ctagggcccc cggggcgtat   61gacgccggag ccctctgacc gcacctctga ccacaacaaa cccctactcc acccgtcttg  121tttgtcccac ccttggtgac gcagagcccc agcccagacc ccgcccaaag cactcattta  181actggtattg cggagccacg aggcttctgc ttactgcaac tcgctccggc cgctgggcgt  241agctgcgact cggcggagtc ccggcggcgc gtccttgttc taacccggcg cgccatgacc  301gtcgcgcggc cgagcgtgcc cgcggcgctg cccctcctcg gggagctgcc ccggctgctg  361ctgctggtgc tgttgtgcct gccggccgtg tggggt gact gtggccttcc cccagatgta  421cctaatgccc agccagcttt ggaaggccgt acaagttttc ccgaggatac tgtaataacg  481tacaaatgtg aagaaagctt tgtgaaaatt cctggcgaga aggactcagt gatctgcctt  541aagggcagtc aatggtcaga tattgaagag ttctgcaatc gtagctgcga ggtgccaaca  601aggctaaatt ctgcatccct caaacagcct tatatcactc agaattattt tccagtcggt  661actgttgtgg aatatgagtg ccgtccaggt tacagaagag aaccttctct atcaccaaaa  721ctaacttgcc ttcagaattt aaaatggtcc acagcagtcg aattttgtaa aaagaaatca  781tgccctaatc cgggagaaat acgaaatggt cagattgatg taccaggtgg catattattt  841ggtgcaacca tctccttctc atgtaacaca gggtacaaat tatttggctc gacttctagt  901ttttgtctta tttcaggcag ctctgtccag tggagtgacc cgttgccaga gtgcagagaa  961atttattgtc cagcaccacc acaaattgac aatggaataa ttcaagggga acgtgaccat 1021tatggatata gacagtctgt aacgtatgca tgtaataaag gattcaccat gattggagag 1081cactctattt attgtactgt gaataatgat gaaggagagt ggagtggccc accacctgaa 1141tgcagaggaa aatctctaac ttccaaggtc ccaccaacag ttcagaaacc taccacagta 1201aatgttccaa ctacagaagt ctcaccaact tctcagaaaa ccaccacaaa aaccaccaca 1261ccaaatgctc aagcaacacg gagtacacct gtttccagga caaccaagca ttttcatgaa 1321acaaccccaa ataaaggaag tggaaccact tcaggtacta cccgtcttct atctgggcac 1381acgtgtttca cgttgacagg tttgcttggg acgctagtaa ccatgggctt gctgacttag 1441ccaaagaaga gttaagaaga aaatacacac aagtatacag actgttccta gtttcttaga.

Example 9. Exemplary HLA-Cw*3 and 5 Sequences

One or more of the following sequence may be used to identify and/oridentify immune cells express TCR sequences specific for HLA-Cw*3 and 5.

Epitope SEQ ID Epitope SEQ ID ID Sequence NO: ID Sequence NO:    515AAYAAAAAL 9 224793 IPNEIIHAL 134   5714 AYAAQGYKVL 10 229882 AAVDAGMAM135  15298 FAVPNLQSL 11 229895 FAYDGKDYLTL 136  17619 FRNLAYGRTCVLGK 12229910 LAVHPSGVAL 137  17892 FSYMDDVVL 13 420580 AAAAAAAAL 138  18044FTPPHGGLL 14 420581 AAYDAAAAA 139  18328 FVYGGSKTSL 15 420582AAYDAAAAAAAL 140  33104 KQYLNLYPV 16 420583 AAYDAAAAAAL 141  42946MVHQAISPR 17 420584 AAYDAAAAAL 142  50089 PYFVRAQGLI 18 420585 AAYDAAAAL143  51649 QMVHQAISPR 19 420586 AAYDAAAL 144  69922 VMAPRTLVL 20 420595FAYDGKDYIAL 145  70932 VSFIEFVGW 21 420597 GAVDPLKAL 146  78969FAYDGKDYI 22 420615 IAAGIFNDL 147  78970 FAYDGKDYL 23 420616 IAIDPSKKL148 114817 FAMPNFQTL 24 420617 IAIIPSKAL 149 141601 AAADAAAAL 25 420618IAIIPSKKL 150 141649 GAVDPLLAL 26 420651 PAAAAFDL 151 141650 GAVDPLLKL27 420660 TAMDVVYKL 152 141651 GAVDPLLSL 28 420665 VAYEAPSL 153 141652GAVDPLLVL 29 433086 VTDSGPTFNYL 154 141653 GAVDPLLYL 30 436040 AAAYPHTSL155 141727 QAISPRTL 31 436053 AAMKALQAL 156 141759 TAMDVVYAL 32 436062AAVDRTTEL 157 144186 AAMPNFQTA 33 436063 AAVPRAAFL 158 144187 AAMPNFQTL34 436324 ALAPGVRAV 159 144209 AQAISPRTL 35 436332 ALFQHITAL 160 144259FAAISPRTL 36 436362 ALNPVANFHL 161 144260 FAAPNFQTA 37 436704 AVMLHSFTL162 144261 FAAPNFQTL 38 437182 FAALHGPAL 163 144262 FAGISPRTL 39 437183FADGHVLEL 164 144263 FAMANFQTA 40 437185 FADPHGKVF 165 144264 FAMANFQTL41 437186 FAEDSLRVI 166 144265 FAMPAFQTA 42 437187 FAEGFVRAL 167 144266FAMPAFQTL 43 437188 FAEGQGRAL 168 144267 FAMPNAQTA 44 437190 FAGPHSTL169 144268 FAMPNAQTL 45 437191 FAGWGRAL 170 144269 FAMPNFATA 46 437192FAIDPHLLL 171 144270 FAMPNFATL 47 437195 FAMIPAKEA 172 144271 FAMPNFQAA48 437200 FASELSRSL 173 144272 FAMPNFQAL 49 437204 FAVIAHVGM 174 144273FAMPNFQTA 50 437205 FAVISRHSL 175 144274 FAMPNLQTA 51 437206 FAYEGTRDSGM176 144275 FAMPNLQTL 52 437258 FGIDRPAEL 177 144283 FGMPNFQTA 53 437268FGQLGREL 178 144284 FGMPNFQTL 54 437272 FIEDAVHVL 179 144285 FGMPNFQTM55 437303 FLADNHHQV 180 144291 FQAISPRTL 56 437309 FLDEKTHEL 181 144295FVMPNFQTA 57 437349 FNDPNAKEM 182 144296 FVMPNFQTL 58 437350 FNFVGKIL183 144313 GQMVHQAI 59 437421 FQHVGQTL 184 144314 GQMVHQAIS 60 437446FSHPREPAL 185 144315 GQMVHQAISPRT 61 437447 FSKIGGIL 186 144316GQMVHQAISPRTL 62 437451 FSSANSPFL 187 144327 HAAISPRTL 63 437463FTVPHTHVF 188 144328 HAGISPRTL 64 437472 FVIETARQL 189 144329 HAMPNFQTA65 437478 FVNPHVSSF 190 144330 HAMPNFQTL 66 437629 GIHETTFNSIM 191144332 HQAASPRTL 67 437771 GQYPRALEL 192 144333 HQAIGPRTL 68 437882HAFLKTEF 193 144334 HQAISARTL 69 437893 HAYLSKNSL 194 144335 HQAISPATL70 438072 IAAPFTSKL 195 144336 HQAISPRAL 71 438083 IAQPVRSFL 196 144337HQAISPRT 72 438087 IATYRTLL 197 144338 HQAISPRTA 73 438206 ILQPHVIAL 198144339 HQAISPRTD 74 438212 IMNDIPIRL 199 144340 HQAISPRTF 75 438292IQDDMHLVI 200 144341 HQAISPRTK 76 438328 ISEENFRVM 201 144342 HQAISPRTL77 438377 IVIDPKNPL 202 144343 HQAISPRTM 78 438413 KAFDYPSRF 203 144344HQDISPRTL 79 438418 KAHLGTAL 204 144345 HQGISPRTL 80 438426 KAWENSPNV205 144346 HQKISPRTL 81 438602 KIAPNTPQL 206 144347 HQLISPRTL 82 438718KMWENRQNL 207 144348 HQPISPRTL 83 438791 KQMEQISQFL 208 144349 HQSISPRTL84 438850 KVAPAPAVV 209 144389 LAMPNFQTA 85 438868 KVIDGLETL 210 144390LAMPNFQTL 86 438900 KVYERAVEF 211 144416 MVHQAISPRT 87 438922 LADPVFRTL212 144447 QMVHQAIS 88 438928 LALTRSSSL 213 144521 VHQAISPR 89 438930LAMRPLASL 214 144522 VHQAISPRT 90 438934 LAVDKSASL 215 144523 VHQAISPRTL91 438936 LAYPARPAQL 216 156625 AQFEHTILL 92 439019 LIVEPSREL 217 162246FQNPFRSEL 93 439272 MADPNIRFL 218 162775 KSMETKVQF 94 439413 MVIVPTREL219 163272 RLYPEGLAQL 95 439545 NQYAYDGKDYIAL 220 N 163894 YAYDGKDYIAL96 439773 RAFPYGNVAF 221 190418 FSSAGPCAL 97 440002 RLFESSQYL 222 190421FTGLYSSTV 98 440047 RLYQGINQL 223 190422 FTQCGYPAL 99 440302 RVWDAEHPGL224 190423 FTSAICSVV 100 440326 SAFDHFASV 225 190438 GTFVSPLPI 101440334 SAIGRAMEL 226 190457 KSVQHLESL 102 440335 SAIGYIHSL 227 190477LQDPRVRAL 103 440336 SAIPHPLIM 228 190480 LSLDVSAAF 104 440346 SAYERSMM229 190481 LSPTVWLSV 105 441161 TQLGPPYHIL 230 190485 LSYQHFRKL 106441233 VADTVARVL 231 190492 MAARLCCQL 107 441235 VAEESRQVL 232 190497MMWFWGPSL 108 441246 VAMGYSHSL 233 190526 RAFPHCLAF 109 441253 VAYLQAHAL234 190536 SAAFYHLPL 110 441346 VGDPSVHLL 235 190581 VSIPWTHKV 111441385 VIYPARISL 236 190592 YAAVTNFLL 112 441552 VSQVGKEL 237 191716RVAPEEHPVL 113 441562 VTDPTGFLRM 238 193448 FVYGJSKTSL + 114 441593VTVRPGLAM 239 MCM(X5) 193682 ALRDVSEEL 115 441621 VVMNVVHQL 240 193740FLAEHPNVTL 116 441622 VVMPIAHEF 241 193751 FLDKNDHSL 117 441635VVVAVGRAL 242 193814 FLSEHPNVTL 118 441687 YADPTKRLEL 243 194187LLYQGPHNTL 119 441689 YAEVGRVL 244 194188 LMAEMGVHSV 120 441694YAMDYSNKAL 245 194305 RMLDSVEKL 121 441702 YAYDGKDYLAL 246 194312RVPPPPQSV 122 441812 YGTRYGASL 247 194557 YAYEKPHVV 123 441815 YGYGHESEL248 194656 YMYEKESEL 124 441949 YQMEKDIAM 249 195533 ISLEGKPL 125 441976YSDDIPHAL 250 196459 FIAPTGHSL 126 441980 YSHDGAFL 251 196711 NTIDPSHPM127 441987 YSLDHISSL 252 208210 FAHPYQYEL 128 441989 YSNRVVDL 253 220053TGYIKTEL 129 442007 YTYTSKAL 254 220571 VATEGSREL 130 461955 AVRDLERAM255 221631 YGFEKPSAI 131 474614 AATATFAAA 256 221641 YGNPRTNGM 132478229 FSIDSPDSL 257 224734 FTDEESRVF 133 488584 VVPEPGQPL 258

REFERENCES

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Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. A ghost cell trainer (GHT), wherein said GHT comprises a plasmamembrane having: a. a plurality of MEW molecules; and b. a plurality ofa protein of interest, wherein the protein isglycosylphosphatidylinositol (GPI) anchored on the cell surface.
 2. Amethod of treating a subject having cancer, the method comprising: a.transfecting a cell expressing a major histocompatibility complex (MHC)molecule with a plurality of vectors each comprising a gene encoding aprotein of interest and a glycosylphosphatidylinositol (GPI) signalinganchor, or a vector comprising a fusion gene encoding a plurality ofproteins of interest and GPI signaling anchors, such that the proteinsof interest are expressed and anchored to the cell surface via the GPIsignaling anchors, wherein the proteins of interest include IL-15,IL-21, IL-2, CD86, CD64, and CD137L; b. lysing the cell of step (a) byhypotonic means to form a ghost cell (GHC) that is substantially free ofall intracellular components; c. co-culturing the GHC with an immunecell isolated from the subject to expand and activate the immune cell,wherein the immune cell is a B cell; and d. infusing the activatedimmune cell into the subject.
 3. The method of claim 2, wherein the GPIanchored proteins of interest are within a lipid raft.
 4. The method ofclaim 2, wherein the MEW molecule is HLA-Cw*3 and/or HLA-Cw*5.
 5. Themethod of claim 2, wherein said GHC is of human, canine, feline, murine,or equine origin.
 6. The method of claim 2, wherein said GHC is derivedfrom a primary cell culture or an immortalized cell line.
 7. The methodof claim 6, wherein the immortalized cell line is K562 or Jurkat.
 8. Themethod of claim 2, wherein the cancer is a hematologic cancer or a solidcancer.
 9. The method of claim 8, wherein the hematologic cancer isleukemia or lymphoma.
 10. The method of claim 8, wherein the solidcancer is osteosarcoma, hemangiosarcoma, transitional cell carcinoma,melanoma, glioblastoma, neuroblastoma, mammary carcinoma, or a sarcomaor carcinoma of the gastrointestinal system.
 11. The method of claim 2,wherein the subject is a canine, feline, or equine.
 12. A method oftreating a subject having cancer, the method comprising: a. transfectinga cell with i. a vector encoding a major histocompatibility complex(MHC) molecule and a glycosylphosphatidylinositol (GPI) signalinganchor; and ii. a plurality of vectors each comprising a gene encoding aprotein of interest and a GPI signaling anchor, or a vector comprising afusion gene encoding a plurality of proteins of interest and GPIsignaling anchors, such that the MHC molecule and the proteins ofinterest are expressed and anchored to the cell surface via the GPIsignaling anchors, wherein the proteins of interest include IL-15,IL-21, IL-2, CD86, CD64, and CD137L; b. lysing the cell of step (a) byhypotonic means to form a ghost cell (GHC) that is substantially free ofall intracellular components; c. co-culturing the GHC with an immunecell isolated from the subject to expand and activate the immune cell,wherein the immune cell is a B cell; and d. infusing the activatedimmune cell into the subject.
 13. The method of claim 12, wherein saidGHC is of human, canine, feline, murine, or equine origin.
 14. Themethod of claim 12, wherein the proteins of interest are within a lipidraft.
 15. The method of claim 12, wherein the WIC molecules are GPIanchored on the cell surface within a lipid raft.
 16. The method ofclaim 12, wherein the WIC molecules are HLA-Cw*3 and/or HLA-Cw*5. 17.The method of claim 12, wherein said GHC is derived from a primary cellculture or an immortalized cell line.
 18. The method of claim 17,wherein the immortalized cell line is K562 or Jurkat.
 19. The method ofclaim 12, wherein the cancer is leukemia, lymphoma, osteosarcoma,hemangiosarcoma, transitional cell carcinoma, melanoma, glioblastoma,neuroblastoma, mammary carcinoma, or a sarcoma or carcinoma of thegastrointestinal system.
 20. The method of claim 12, wherein the subjectis a canine, feline, or equine.